Compositions and methods for inhibiting the proliferation of cells

ABSTRACT

The present invention relates to chemical compounds, methods for their discovery, and their therapeutic use. In particular, the present invention provides benzodiazepine derivatives and methods of using benzodiazepine derivatives as therapeutic agents to treat a number of conditions associated with the faulty regulation of the processes of programmed cell death, autoimmunity, inflammation, and hyperproliferation, and the like.

This application is a continuation of pending U.S. patent applicationSer. No. 11/643,614, filed Dec. 21, 2006, which is a Divisional ofallowed U.S. patent application Ser. No. 10/427,211, filed May 1, 2003(now U.S. Pat. No. 7,572,788), which is a Continuation-in-Part ofabandoned U.S. patent application Ser. No. 10/217,878, filed Aug. 13,2002, which is a Continuation of allowed U.S. patent application Ser.No. 09/767,283, filed Jan. 22, 2001 (now U.S. Pat. No. 7,220,739), whichis a Continuation of allowed U.S. patent application Ser. No.09/700,101, filed Nov. 8, 2000 (now U.S. Pat. No. 7,125,866), which isthe National entry of expired International Patent Application No.PCT/US00/11599 filed Apr. 27, 2000, which claims priority to U.S.Provisional Application No. 60/131,761, filed Apr. 30, 1999, to U.S.Provisional Application No. 60/165,511, filed Nov. 15, 1999, and to U.S.Provisional Application No. 60/191,855, filed Mar. 24, 2000. AbandonedU.S. application Ser. No. 10/217,878 also claims priority to U.S.Provisional Application No. 60/312,560, filed Aug. 15, 2001, to U.S.Provisional Application No. 60/313,689, filed Aug. 20, 2001, and to U.S.Provisional Application No. 60/396,670, filed Jul. 18, 2002. Eachaforementioned application is specifically incorporated herein byreference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made in part with government support under Grant Nos.GM46831 and AI47450 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to novel chemical compounds, methods fortheir discovery, and their therapeutic use. In particular, the presentinvention provides benzodiazepine derivatives and related compounds andmethods of using benzodiazepine derivatives and related compounds astherapeutic agents to treat a number of conditions associated with thefaulty regulation of the processes of programmed cell death,autoimmunity, inflammation, hyperproliferation, and the like.

BACKGROUND OF THE INVENTION

Multicellular organisms exert precise control over cell number. Abalance between cell proliferation and cell death achieves thishomeostasis. Cell death occurs in nearly every type of vertebrate cellvia necrosis or through a suicidal form of cell death, known asapoptosis. Apoptosis is triggered by a variety of extracellular andintracellular signals that engage a common, genetically programmed deathmechanism.

Multicellular organisms use apoptosis to instruct damaged or unnecessarycells to destroy themselves for the good of the organism. Control of theapoptotic process therefore is very important to normal development, forexample, fetal development of fingers and toes requires the controlledremoval, by apoptosis, of excess interconnecting tissues, as does theformation of neural synapses within the brain. Similarly, controlledapoptosis is responsible for the sloughing off of the inner lining ofthe uterus (the endometrium) at the start of menstruation. Whileapoptosis plays an important role in tissue sculpting and normalcellular maintenance, it is also the primary defense against cells andinvaders (e.g., viruses) which threaten the well being of the organism.

Not surprisingly many diseases are associated with dysregulation of theprocess of cell death. Experimental models have established acause-effect relationship between aberrant apoptotic regulation and thepathenogenicity of various neoplastic, autoimmune and viral diseases.For instance, in the cell mediated immune response, effector cells(e.g., cytotoxic T lymphocytes “CTLs”) destroy virus-infected cells byinducing the infected cells to undergo apoptosis. The organismsubsequently relies on the apoptotic process to destroy the effectorcells when they are no longer needed. Autoimmunity is normally preventedby the CTLs inducing apoptosis in each other and even in themselves.Defects in this process are associated with a variety of autoimmunediseases such as lupus erythematosus and rheumatoid arthritis.

Multicellular organisms also use apoptosis to instruct cells withdamaged nucleic acids (e.g., DNA) to destroy themselves prior tobecoming cancerous. Some cancer-causing viruses overcome this safeguardby reprogramming infected (transformed) cells to abort the normalapoptotic process. For example, several human papilloma viruses (HPVs)have been implicated in causing cervical cancer by suppressing theapoptotic removal of transformed cells by producing a protein (E6) whichinactivates the p53 apoptosis promoter. Similarly, the Epstein-Barrvirus (EBV), the causative agent of mononucleosis and Burkitt'slymphoma, reprograms infected cells to produce proteins that preventnormal apoptotic removal of the aberrant cells thus allowing thecancerous cells to proliferate and to spread throughout the organism.

Still other viruses destructively manipulate a cell's apoptoticmachinery without directly resulting in the development of a cancer. Forexample, the destruction of the immune system in individuals infectedwith the human immunodeficiency virus (HIV) is thought to progressthrough infected CD4⁺ T cells (about 1 in 100,000) instructinguninfected sister cells to undergo apoptosis.

Some cancers that arise by non-viral means have also developedmechanisms to escape destruction by apoptosis. Melanoma cells, forinstance, avoid apoptosis by inhibiting the expression of the geneencoding Apaf-1. Other cancer cells, especially lung and colon cancercells, secrete high levels of soluble decoy molecules that inhibit theinitiation of CTL mediated clearance of aberrant cells. Faultyregulation of the apoptotic machinery has also been implicated invarious degenerative conditions and vascular diseases.

It is apparent that the controlled regulation of the apoptotic processand its cellular machinery is vital to the survival of multicellularorganisms. Typically, the biochemical changes that occur in a cellinstructed to undergo apoptosis occur in an orderly procession. However,as shown above, flawed regulation of apoptosis can cause seriousdeleterious effects in the organism.

There have been various attempts to control and restore regulation ofthe apoptotic machinery in aberrant cells (e.g., cancer cells). Forexample, much work has been done to develop cytotoxic agents to destroyaberrant cells before they proliferate. As such, cytotoxic agents havewidespread utility in both human and animal health and represent thefirst line of treatment for nearly all forms of cancer andhyperproliferative autoimmune disorders like lupus erythematosus andrheumatoid arthritis. Many cytotoxic agents in clinical use exert theireffect by damaging DNA (e.g., cis-diaminodichroplatanim(II) cross-linksDNA, whereas bleomycin induces strand cleavage). The result of thisnuclear damage, if recognized by cellular factors like the p53 system,is to initiate an apoptotic cascade leading to the death of the damagedcell.

However, existing cytotoxic chemotherapeutic agents have seriousdrawbacks. For example, many known cytotoxic agents show littlediscrimination between healthy and diseased cells. This lack ofspecificity often results in severe side effects that can limit efficacyand/or result in early mortality. Moreover, prolonged administration ofmany existing cytotoxic agents results in the expression of resistancegenes (e.g., bcl-2 family or multi-drug resistance (MDR) proteins) thatrender further dosing either less effective or useless. Some cytotoxicagents induce mutations into p53 and related proteins. Based on theseconsiderations, ideal cytotoxic drugs should only kill diseased cellsand not be susceptible to chemo-resistance.

One strategy to selectively kill diseased cells is to develop drugs thatselectively recognize molecules expressed in diseased cells. Thus,effective cytotoxic chemotherapeutic agents, would recognize diseaseindicative molecules and induce (e.g., either directly or indirectly)the death of the diseased cell. Although markers on some types of cancercells have been identified and targeted with therapeutic antibodies andsmall molecules, unique traits for diagnostic and therapeuticexploitation are not known for most cancers. Moreover, for diseases likelupus, specific molecular targets for drug development have not beenidentified.

What are needed are improved compositions and methods for regulating theapoptotic processes in subjects afflicted with diseases and conditionscharacterized by faulty regulation of these processes (e.g., viralinfections, hyperproliferative autoimmune disorders, chronicinflammatory conditions, and cancers).

SUMMARY

The present invention provides novel compounds that find use in treatinga number of diseases and conditions and that find use in research,compound screening, and diagnostic applications. The present inventionalso provides uses of these novel compounds, as well as the use of knowncompounds, that elicit particular biological responses (e.g., compoundsthat bind to particular target molecules and/or cause particularcellular events). Such compounds and uses are described throughout thepresent application and represent a diverse collection of compositionsand applications.

Certain preferred compositions and uses are described below. The presentinvention is not limited to these particular compositions and uses.

The present invention provides a number of useful compositions asdescribed throughout the present application. Certain preferredembodiments of the present involve compositions include a compositioncomprising the following formula:

wherein R₁ is selected from napthalalanine; phenol; 1-Napthalenol;2-Napthalenol;

and quinolines; wherein R₂ is selected from the group consisting of:

and wherein R₁ and R₂ include both R or S enantiomeric forms and racemicmixtures. .

Other preferred embodiments of the present involve compositions includea composition comprising the following formula:

wherein R1 is selected from H, alkyl, or substituted alkyl; wherein R2is selected from hydrogen, a hydroxy, an slkoxy, a halo, an amino, alower-alkyl, a substituted amino, an acetylamino, a hydroxyamino, analiphatic group having 1-8 carbons and 1-20 hydrogens, a substitutedaliphatic group of similar size, a cycloaliphatic group consisting of<10 carbons, a substituted cycloaliphatic group, an aryl, aheterocyclic; wherein R3 is selected from H, alkyl, or substitutedalkyl, and wherein at most one substituent is a hydroxyl subgroup;wherein R4 is selected from

wherein n=0-5; and wherein R₁, R₂, R₃ and R₄ include both R or Senantiomeric forms and racemic mixtures.

Still other preferred embodiments of the present involve compositionsinclude a composition comprising the following formula:

wherein R1 is selected from H, alkyl, or substituted alkyl; wherein R2is selected from hydrogen, a hydroxy, an alkoxy, a halo, an amino, alower-alkyl, a substituted amino, an acetylamino, a hydroxyamino, analiphatic group having 1-8 carbons and 1-20 hydrogens, a substitutedaliphatic group of similar size, a cycloaliphatic group consisting of<10 carbons, a substituted cycloaliphatic group, an aryl, aheterocyclic; wherein R3 is selected from H, alkyl, or substitutedalkyl, and wherein at most one substituent is a hydroxyl subgroup;wherein R4 is selected from

wherein n=0-5; and wherein R₁, R₂, R₃ and R₄ include both R or Senantiomeric forms and racemic mixtures.

In other preferred embodiments, the present invention provides apharmaceutical composition. In such embodiments, the present inventionprovides a compound that binds to oligomycin conferring protein, and anagent (e.g., resveratrol, picetannol, estrogen, lansoprazole).

The present invention also provides methods and compositions useful inregulating cellular death. In preferred embodiments, the presentinvention provides a subject and a composition comprising a formulaselected from the group consisting of

wherein R is selected from hydrogen, a hydroxy, an alkoxy, a halo, anamino, a lower-alkyl-a substituted-amino, an acetylamino, ahydroxyamino, an aliphatic group having 1-8 carbons and 1-20 hydrogens,a substituted aliphatic group of similar size, a cycloaliphatic groupconsisting of <10 carbons, a substituted cycloaliphatic group, an aryl,and a heterocyclic; and such a composition is administered to thesubject.

In still other preferred embodiments, the present invention providescompositions and methods for regulating cellular proliferation. In suchembodiments, the present invention provides a subject and a compositioncomprising a formula selected from

wherein R is selected from hydrogen, a hydroxy, an alkoxy, a halo, anamino, a lower-alkyl-a substituted-amino, an acetylamino, ahydroxyamino, an aliphatic group having 1-8 carbons and 1-20 hydrogens,a substituted aliphatic group of similar size, a cycloaliphatic groupconsisting of <10 carbons, a substituted cycloaliphatic group, an aryl,and a heterocyclic; and the compostion is administered to the subject.

The present invention provides a number of methods for influencing thefate of cells, tissues, and organisms. Certain preferred embodiments ofthe present involve methods for regulating cell death. In suchembodiments, the present invention provides target cells havingmitochondria and a composition comprising the following formula:

wherein R1 comprises a hydrophobic aromatic group larger than benzene;wherein R2 comprises a phenolic hydroxyl group; and wherein R₁, and R₂include both R or S enantiomeric forms and racemic mixtures. Inadditional embodiments, the cells are exposed to the composition underconditions such that said composition binds to the oligomycinsensitivity conferring protein so as to increase superoxide levels oralter cellular ATP levels in said cells.

In other embodiments, target cells are in vitro cells. In otherembodiments, the target cells are in vivo cells. In still otherembodiments, the target cells are ex vivo cells. In yet otherembodiments, the target cells are cancer cells. In some embodiments, thetarget cells are selected from the group consisting of B cells, T cells,and granulocytes.

In other embodiments used in the regulation of cellular death, thepresent invention also provides the following compositions:

wherein R₁ is selected from group consisting of: napthalalanine; phenol;1-Napthalenol; 2-Napthalenol;

and quinolines; wherein R₂ is selected from the group consisting of:

and wherein R₁ and R₂ include both R or S enantiomeric forms and racemicmixtures.

In preferred embodiments wherein the present invention regulatescellular death, exposure of the composition to target cells results inan increase in cell death of the target cells.

The present invention also provides methods and compositions forregulating cellular proliferation. In such embodiments, the presentinvention provides proliferating target cells having mitochondria, and acomposition comprising the following formula:

wherein comprises a hydrophobic aromatic group larger than benzene;wherein R2 comprises a phenolic hydroxyl group; wherein R₁ and R₂include both R or S enantiomeric forms and racemic mixtures; and whereinthe cells are exposed to the composition under conditions such that thecomposition binds to the mitochondrial ATP synthase complex so as toincrease superoxide levels or alter cellular ATP levels in the cells. Inpreferred embodiments, the composition binds to oligomycin sensitivityconferring protein.

In some embodiments, the target cells are in vitro cells. In otherembodiments, the target cells are in vivo cells. In still otherembodiments, the target cells are ex vivo cells. In other embodiments,the target cells are cancer cells. In yet other embodiments, the targetcells are selected from the group consisting of B cells, T cells, andgranulocytes. In still further embodiments, the target cells areproliferating cells.

In other embodiments wherein the present invention regulates cellularproliferation, the present invention provides the following composition:

wherein R₁ is selected from napthalalanine; phenol; 1-Napthalenol;2-Napthalenol;

and quinolines; wherein R₂ is selected from the group consisting of:

DESCRIPTION OF THE FIGURES

FIG. 1 shows data demonstrating that the OSCP component is a bindingprotein for Bz-423. Semiquantitative PCR using primers specific for theOSCP sequence reveals enrichment of clones encoding the OSCP with eachround of selection using immobilized Bz-423.

FIG. 2 is a graph showing the binding isotherm of Bz-423 and purifiedhuman OSCP.

FIG. 3 shows siRNA regulation of OSCP. 10⁶ HEK 293 cells transfectedwith 0.84 μg of siRNA duplexes specific for OSCP or scrambled control.After 72 h, protein expression determined by immunoblotting whole cellextracts. OSCP is reduced and GAPDH increased as glycolytic response.

FIG. 4 shows data showing gene expression profiles of cells treated bythe compounds of the present invention. Data from an expression analysisfor genes up-regulated in the presence of Bz-423 is presented in FIG.4A-1 to 4A-9. Data from an expression analysis for genes down-regulatedin the presence of Bz-423 is presented in FIG. 4B-1 to 4B-14. Data froman expression analysis for genes up-regulated in the presence of Bz-OMeis presented in FIG. 4C-1 to 4C-16. Data from an expression analysis forgenes down-regulated in the presence of Bz-OMe is presented in FIG. 4D-1to 4D-13.

DEFINITIONS

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below.

As used herein, the term “benzodiazepine” refers to a seven memberednon-aromatic heterocyclic ring fused to a phenyl ring wherein theseven-membered ring has two nitrogen atoms, as part of the heterocyclicring. In some aspects, the two nitrogen atoms are in 1 and 4 positions,as shown in the general structure below.

The benzodiazepine can be substituted with one keto group (typically atthe 2-position), or with two keto groups, one each at the 2- and 5-positions. When the benzodiazepine has two keto groups, one each at the2- and 5-positions, it is referred to as benzodiazepine-2,5-dione. Mostgenerally, the benzodiazepine is further substituted either on thesix-membered phenyl ring or on the seven-membered heterocyclic ring oron both rings by a variety of substituents. These substituents aredescribed more fully herein. The term “larger than benzene” refers toany chemical group containing 7 or more non-hydrogen atoms.

As used herein, the term “substituted aliphatic” refers to an alkanepossessing less than 10 carbons where at least one of the aliphatichydrogen atoms has been replaced by a halogen, an amino, a hydroxy, anitro, a thio, a ketone, an aldehyde, an ester, an amide, a loweraliphatic, a substituted lower aliphatic, or a ring (aryl, substitutedaryl, cycloaliphatic, or substituted cycloaliphatic, etc.). Examples ofsuch include, but are not limited to, 1-chloroethyl and the like.

As used herein, the term “substituted aryl” refers to an aromatic ringor fused aromatic ring system consisting of no more than three fusedrings at least one of which is aromatic, and where at least one of thehydrogen atoms on a ring carbon has been replaced by a halogen, anamino, a hydroxy, a nitro, a thio, a ketone, an aldehyde, an ester, anamide, a lower aliphatic, a substituted lower aliphatic, or a ring(aryl, substituted aryl, cycloaliphatic, or substituted cycloaliphatic).Examples of such include, but are not limited to, hydroxyphenyl and thelike.

As used herein, the term “cycloaliphatic” refers to a cycloalkanepossessing less than 8 carbons or a fused ring system consisting of nomore than three fused cycloaliphatic rings. Examples of such include,but are not limited to, decalin and the like.

As used herein, the term “substituted cycloaliphatic” refers to acycloalkane possessing less than 8 carbons or a fused ring systemconsisting of no more than three fused rings, and where at least one ofthe aliphatic hydrogen atoms has been replaced by a halogen, a nitro, athio, an amino, a hydroxy, a ketone, an aldehyde, an ester, an amide, alower aliphatic, a substituted lower aliphatic, or a ring (aryl,substituted aryl, cycloaliphatic, or substituted cycloaliphatic).Examples of such include, but are not limited to, 1-chlorodecalyl andthe like.

As used herein, the term “heterocyclic” refers to a cycloalkane and/oran aryl ring system, possessing less than 8 carbons, or a fused ringsystem consisting of no more than three fused rings, where at least oneof the ring carbon atoms is replaced by oxygen, nitrogen or sulfur.Examples of such include, but are not limited to, morpholino and thelike.

As used herein, the term “substituted heterocyclic” refers to acycloalkane and/or an aryl ring system, possessing less than 8 carbons,or a fused ring system consisting of no more than three fused rings,where at least one of the ring carbon atoms is replaced by oxygen,nitrogen or sulfur, and where at least one of the aliphatic hydrogenatoms has been replaced by a halogen, hydroxy, a thio, nitro, an amino,a ketone, an aldehyde, an ester, an amide, a lower aliphatic, asubstituted lower aliphatic, or a ring (aryl, substituted aryl,cycloaliphatic, or substituted cycloaliphatic). Examples of suchinclude, but are not limited to 2-chloropyranyl.

As used herein, the term “linker” refers to a chain containing up to andincluding eight contiguous atoms connecting two different structuralmoieties where such atoms are, for example, carbon, nitrogen, oxygen, orsulfur. Ethylene glycol is one non-limiting example.

As used herein, the term “lower-alkyl-substituted-amino” refers to anyalkyl unit containing up to and including eight carbon atoms where oneof the aliphatic hydrogen atoms is replaced by an amino group. Examplesof such include, but are not limited to, ethylamino and the like.

As used herein, the term “lower-alkyl-substituted-halogen” refers to anyalkyl chain containing up to and including eight carbon atoms where oneof the aliphatic hydrogen atoms is replaced by a halogen. Examples ofsuch include, but are not limited to, chlorethyl and the like.

As used herein, the term “acetylamino” shall mean any primary orsecondary amino that is acetylated. Examples of such include, but arenot limited to, acetamide and the like.

The term “derivative” of a compound, as used herein, refers to achemically modified compound wherein the chemical modification takesplace either at a functional group of the compound or on the aromaticring. Non-limiting examples of 1,4-benzodiazepine derivatives of thepresent invention may include N-acetyl, N-methyl, N-hydroxy groups atany of the available nitrogens in the compound. Additional derivativesmay include those having a trifluoromethyl group on the phenyl ring.

As used herein, the term “subject” refers to organisms to be treated bythe methods of the present invention. Such organisms preferably include,but are not limited to, mammals (e.g., murines, simians, equines,bovines, porcines, canines, felines, and the like), and most preferablyincludes humans. In the context of the invention, the term “subject”generally refers to an individual who will receive or who has receivedtreatment (e.g., administration of benzodiazepine compound(s), andoptionally one or more other agents) for a condition characterized bythe dysregulation of apoptotic processes.

The term “diagnosed,” as used herein, refers to the to recognition of adisease by its signs and symptoms (e.g., resistance to conventionaltherapies), or genetic analysis, pathological analysis, histologicalanalysis, and the like.

As used herein, the terms “anticancer agent,” or “conventionalanticancer agent” refer to any chemotherapeutic compounds, radiationtherapies, or surgical interventions, used in the treatment of cancer.

As used herein the term, “in vitro” refers to an artificial environmentand to processes or reactions that occur within an artificialenvironment. In vitro environments include, but are not limited to, testtubes and cell cultures. The term “in vivo” refers to the naturalenvironment (e.g., an animal or a cell) and to processes or reactionthat occur within a natural environment.

As used herein, the term “host cell” refers to any eukaryotic orprokaryotic cell (e.g., mammalian cells, avian cells, amphibian cells,plant cells, fish cells, and insect cells), whether located in vitro orin vivo.

As used herein, the term “cell culture” refers to any in vitro cultureof cells. Included within this term are continuous cell lines (e.g.,with an immortal phenotype), primary cell cultures, finite cell lines(e.g., non-transformed cells), and any other cell population maintainedin vitro, including oocytes and embryos.

In preferred embodiments, the “target cells” of the compositions andmethods of the present invention include, refer to, but are not limitedto, lymphoid cells or cancer cells. Lymphoid cells include B cells, Tcells, and granulocytes. Granulocyctes include eosinophils andmacrophages. In some embodiments, target cells are continuously culturedcells or uncultered cells obtained from patient biopsies.

Cancer cells include tumor cells, neoplastic cells, malignant cells,metastatic cells, and hyperplastic cells. Neoplastic cells can be benignor malignant. Neoplastic cells are benign if they do not invade ormetastasize. A malignant cell is one that is able to invade and/ormetastasize. Hyperplasia is a pathologic accumulation of cells in atissue or organ, without significant alteration in structure orfunction.

In one specific embodiment, the target cells exhibit pathological growthor proliferation. As used herein, the term “pathologically proliferatingor growing cells” refers to a localized population of proliferatingcells in an animal that is not governed by the usual limitations ofnormal growth.

As used herein, the term “un-activated target cell” refers to a cellthat is either in the G_(o) phase or one in which a stimulus has notbeen applied.

As used herein, the term “activated target lymphoid cell” refers to alymphoid cell that has been primed with an appropriate stimulus to causea signal transduction cascade, or alternatively, a lymphoid cell that isnot in G_(o) phase. Activated lymphoid cells may proliferate, undergoactivation induced cell death, or produce one or more of cytotoxins,cytokines, and other related membrane-associated proteins characteristicof the cell type (e.g., CD8⁺ or CD4⁺). They are also capable ofrecognizing and binding any target cell that displays a particularantigen on its surface, and subsequently releasing its effectormolecules.

As used herein, the term “activated cancer cell” refers to a cancer cellthat has been primed with an appropriate stimulus to cause a signaltransduction. An activated cancer cell may or may not be in the G_(O)phase.

An activating agent is a stimulus that upon interaction with a targetcell results in a signal transduction cascade. Examples of activatingstimuli include, but are not limited to, small molecules, radiantenergy, and molecules that bind to cell activation cell surfacereceptors. Responses induced by activation stimuli can be characterizedby changes in, among others, intracellular Ca²⁺, superoxide, or hydroxylradical levels; the activity of enzymes like kinases or phosphatases; orthe energy state of the cell. For cancer cells, activating agents alsoinclude transforming oncogenes.

In one aspect, the activating agent is any agent that binds to a cellsurface activation receptor. These can be selected from the groupconsisting of a T cell receptor ligand, a B cell activating factor(“BAFF”), a TNF, a Fas ligand (FasL), a CD40 ligand, a proliferationinducing ligand (“APRIL”), a cytokine, a chemokine, a hormone, an aminoacid (e.g., glutamate), a steroid, a B cell receptor ligand, gammairradiation, UV irradiation, an agent or condition that enhances cellstress, or an antibody that specifically recognizes and binds a cellsurface activation receptor (e.g., anti-CD4, anti-CD8, anti-CD20,anti-TACI, anti-BCMA, anti-TNF receptor, anti-CD40, anti-CD3, anti-CD28,anti-B220, anti-CD38, and-CD19, and anti-CD21). BCMA is B cellmaturation antigen receptor and TACI is transmembrane activator and CAMLinteractor. (Gross, A. et al. (2000); Laabi, Y. et al. (1992) and Madry,C. et al. (1998)). Antibodies include monoclonal or polyclonal or amixture thereof.

Examples of a T cell ligand include, but are not limited to, a peptidethat binds to an MHC molecule, a peptide MHC complex, or an antibodythat recognizes components of the T cell receptor.

Examples of a B cell ligand include, but are not limited to, a moleculeor antibody that binds to or recognizes components of the B cellreceptor.

Examples of reagents that bind to a cell surface activation receptorinclude, but are not limited to, the natural ligands of these receptorsor antibodies raised against them (e.g., anti-CD20). RITUXIN (Genentech,Inc., San Francisco, Calif.) is a commercially available anti-CD 20chimeric monoclonal antibody.

Examples of agents or conditions that enhance cell stress include heat,radiation, oxidative stress, or growth factor withdrawal and the like.Examples of growth factors include, but are not limited to serum, IL-2,platelet derived growth factor (“PDGF”), and the like.

As used herein, the term “effective amount” refers to the amount of acompound (e.g., benzodiazepine) sufficient to effect beneficial ordesired results. An effective amount can be administered in one or moreadministrations, applications or dosages and is not limited intended tobe limited to a particular formulation or administration route.

As used herein, the term “dysregulation of the process of cell death”refers to any aberration in the ability of (e.g., predisposition) a cellto undergo cell death via either necrosis or apoptosis. Dysregulation ofcell death is associated with or induced by a variety of conditions,including for example, autoimmune disorders (e.g., systemic lupuserythematosus, rheumatoid arthritis, graft-versus-host disease,myasthenia gravis, Sjogren's syndrome, etc.), chronic inflammatoryconditions (e.g., psoriasis, asthma and Crohn's disease),hyperproliferative disorders (e.g., tumors, B cell lymphomas, T celllymphomas, etc.), viral infections (e.g., herpes, papilloma, HIV), andother conditions such as osteoarthritis and atherosclerosis.

It should be noted that when the dysregulation is induced by orassociated with a viral infection, the viral infection may or may not bedetectable at the time dysregulation occurs or is observed. That is,viral-induced dysregulation can occur even after the disappearance ofsymptoms of viral infection.

A “hyperproliferative disorder,” as used herein refers to any conditionin which a localized population of proliferating cells in an animal isnot governed by the usual limitations of normal growth. Examples ofhyperproliferative disorders include tumors, neoplasms, lymphomas andthe like. A neoplasm is said to be benign if it does not undergo,invasion or metastasis and malignant if it does either of these. Ametastatic cell or tissue means that the cell can invade and destroyneighboring body structures. Hyperplasia is a form of cell proliferationinvolving an increase in cell number in a tissue or organ, withoutsignificant alteration in structure or function. Metaplasia is a form ofcontrolled cell growth in which one type of fully differentiated cellsubstitutes for another type of differentiated cell. Metaplasia canoccur in epithelial or connective tissue cells. A typical metaplasiainvolves a somewhat disorderly metaplastic epithelium.

The pathological growth of activated lymphoid cells often results in anautoimmune disorder or a chronic inflammatory condition. As used herein,the term “autoimmune disorder” refers to any condition in which anorganism produces antibodies or immune cells which recognize theorganism's own molecules, cells or tissues. Non-limiting examples ofautoimmune disorders include rheumatoid arthritis, Sjogren's syndrome,graft versus host disease, myasthenia gravis, systemic lupuserythematosus (“SLE”), and the like.

As used herein, the term “chronic inflammatory condition” refers to acondition wherein the organism's immune cells are activated. Such acondition is characterized by a persistent inflammatory response withpathologic sequelae. This state is characterized by infiltration ofmononuclear cells, proliferation of fibroblasts and small blood vessels,increased connective tissue, and tissue destruction. Examples of chronicinflammatory diseases include, but are not limited to, Crohn's disease,psoriasis, chronic obstructive pulmonary disease, inflammatory boweldisease, multiple sclerosis, and asthma.

Autoimmune diseases such as rheumatoid arthritis and systemic lupuserythematosus can also result in a chronic inflammatory state.

As used herein, the term “co-administration” refers to theadministration of at least two agent(s) (e.g., benzodiazepines) ortherapies to a subject. In some embodiments, the co-administration oftwo or more agents/therapies is concurrent. In other embodiments, afirst agent/therapy is administered prior to a second agent/therapy.Those of skill in the art understand that the formulations and/or routesof administration of the various agents/therapies used may vary. Theappropriate dosage for co-administration can be readily determined byone skilled in the art. In some embodiments, when agents/therapies areco-administered, the respective agents/therapies are administered atlower dosages than appropriate for their administration alone. Thus,co-administration is especially desirable in embodiments where theco-administration of the agents/therapies lowers the requisite dosage ofa known potentially harmful (e.g., toxic) agent(s).

As used herein, the term “toxic” refers to any detrimental or harmfuleffects on a cell or tissue as compared to the same cell or tissue priorto the administration of the toxicant.

As used herein, the term “pharmaceutical composition” refers to thecombination of an active agent with a carrier, inert or active, makingthe composition especially suitable for diagnostic or therapeutic use invivo, in vivo or ex vivo.

As used herein, the term “pharmaceutically acceptable carrier” refers toany of the standard pharmaceutical carriers, such as a phosphatebuffered saline solution, water, emulsions (e.g., such as an oil/wateror water/oil emulsions), and various types of wetting agents. Thecompositions also can include stabilizers and preservatives. Forexamples of carriers, stabilizers and adjuvants. (See e.g., Martin,Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton,Pa. [1975]).

As used herein, the term “pharmaceutically acceptable salt” refers toany pharmaceutically acceptable salt (e.g., acid or base) of a compoundof the present invention which, upon administration to a subject, iscapable of providing a compound of this invention or an activemetabolite or residue thereof As is known to those of skill in the art,“salts” of the compounds of the present invention may be derived frominorganic or organic acids and bases. Examples of acids include, but arenot limited to, hydrochloric, hydrobromic, sulfuric, nitric, perchloric,fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic,toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic,ethanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic,benzenesulfonic acid, and the like. Other acids, such as oxalic, whilenot in themselves pharmaceutically acceptable, may be employed in thepreparation of salts useful as intermediates in obtaining the compoundsof the invention and their pharmaceutically acceptable acid additionsalts.

Examples of bases include, but are not limited to, alkali metals (e.g.,sodium) hydroxides, alkaline earth metals (e.g., magnesium), hydroxides,ammonia, and compounds of formula NW₄ ⁺, wherein W is C₁₋₄ alkyl, andthe like.

Examples of salts include, but are not limited to: acetate, adipate,alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate,citrate, camphorate, camphorsulfonate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, fumarate, flucoheptanoate,glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride,hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmoate,pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate,succinate, tartrate, thiocyanate, tosylate, undecanoate, and the like.Other examples of salts include anions of the compounds of the presentinvention compounded with a suitable cation such as Na⁺, NH₄ ⁺, and NW₄⁺ (wherein W is a C₁₋₄ alkyl group), and the like.

For therapeutic use, salts of the compounds of the present invention arecontemplated as being pharmaceutically acceptable. However, salts ofacids and bases that are non-pharmaceutically acceptable may also finduse, for example, in the preparation or purification of apharmaceutically acceptable compound. As used herein, the terms “solidphase supports” or “solid supports,” are used in their broadest sense torefer to a number of supports that are available and known to those ofordinary skill in the art. Solid phase supports include, but are notlimited to, silica gels, resins, derivatized plastic films, glass beads,cotton, plastic beads, alumina gels, and the like. As used herein,“solid supports” also include synthetic antigen-presenting matrices,cells, liposomes, and the like. A suitable solid phase support may beselected on the basis of desired end use and suitability for variousprotocols. For example, for peptide synthesis, solid phase supports mayrefer to resins such as polystyrene (e.g., PAM-resin obtained fromBachem, Inc., Peninsula Laboratories, etc.), POLYHIPE) resin (obtainedfrom Aminotech, Canada), polyamide resin (obtained from PeninsulaLaboratories), polystyrene resin grafted with polyethylene glycol(TENTAGEL, Rapp Polymere, Tubingen, Germany) or polydimethylacrylamideresin (obtained from Milligen/Biosearch, California).

As used herein, the term “pathogen” refers a biological agent thatcauses a disease state (e.g., infection, cancer, etc.) in a host.“Pathogens” include, but are not limited to, viruses, bacteria, archaea,fungi, protozoans, mycoplasma, prions, and parasitic organisms.

The terms “bacteria” and “bacterium” refer to all prokaryotic organisms,including those within all of the phyla in the Kingdom Procaryotae. Itis intended that the term encompass all microorganisms considered to bebacteria including Mycoplasma, Chlamydia, Actinomyces, Streptomyces, andRickettsia. All forms of bacteria are included within this definitionincluding cocci, bacilli, spirochetes, spheroplasts, protoplasts, etc.Also included within this term are prokaryotic organisms which are gramnegative or gram positive. “Gram negative” and “gram positive” refer tostaining patterns with the Gram-staining process which is well known inthe art. (See e.g., Finegold and Martin, Diagnostic Microbiology, 6thEd., CV Mosby St. Louis, pp. 13-15 [1982]). “Gram positive bacteria” arebacteria which retain the primary dye used in the Gram stain, causingthe stained cells to appear dark blue to purple under the microscope.“Gram negative bacteria” do not retain the primary dye used in the Gramstain, but are stained by the counterstain. Thus, gram negative bacteriaappear red.

As used herein, the term “microorganism” refers to any species or typeof microorganism, including but not limited to, bacteria, archaea,fungi, protozoans, mycoplasma, and parasitic organisms. The presentinvention contemplates that a number of microorganisms encompassedtherein will also be pathogenic to a subject.

As used herein, the term “fungi” is used in reference to eukaryoticorganisms such as the molds and yeasts, including dimorphic fungi.

As used herein, the term “virus” refers to minute infectious agents,which with certain exceptions, are not observable by light microscopy,lack independent metabolism, and are able to replicate only within aliving host cell. The individual particles (i.e., virions) typicallyconsist of nucleic acid and a protein shell or coat; some virions alsohave a lipid containing membrane. The term “virus” encompasses all typesof viruses, including animal, plant, phage, and other viruses.

The term “sample” as used herein is used in its broadest sense. A samplesuspected of indicating a condition characterized by the dysregulationof apoptotic function may comprise a cell, tissue, or fluids,chromosomes isolated from a cell (e.g., a spread of metaphasechromosomes), genomic DNA (in solution or bound to a solid support suchas for Southern blot analysis), RNA (in solution or bound to a solidsupport such as for Northern blot analysis), cDNA (in solution or boundto a solid support) and the like. A sample suspected of containing aprotein may comprise a cell, a portion of a tissue, an extractcontaining one or more proteins and the like.

As used herein, the terms “purified” or “to purify” refer, to theremoval of undesired components from a sample. As used herein, the term“substantially purified” refers to molecules that are at least 60% free,preferably 75% free, and most preferably 90%, or more, free from othercomponents with which they usually associated.

As used herein, the term “antigen binding protein” refers to proteinswhich bind to a specific antigen. “Antigen binding proteins” include,but are not limited to, immunoglobulins, including polyclonal,monoclonal, chimeric, single chain, and humanized antibodies, Fabfragments, F(ab′)2 fragments, and Fab expression libraries. Variousprocedures known in the art are used for the production of polyclonalantibodies. For the production of antibody, various host animals can beimmunized by injection with the peptide corresponding to the desiredepitope including but not limited to rabbits, mice, rats, sheep, goats,etc. In a preferred embodiment, the peptide is conjugated to animmunogenic carrier (e.g., diphtheria toxoid, bovine serum albumin(BSA), or keyhole limpet hemocyanin [KLH]). Various adjuvants are usedto increase the immunological response, depending on the host species,including but not limited to Freund's (complete and incomplete), mineralgels such as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacteriumparvum.

For preparation of monoclonal antibodies, any technique that providesfor the production of antibody molecules by continuous cell lines inculture may be used (See e.g., Harlow and Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).These include, but are not limited to, the hybridoma techniqueoriginally developed by Köhler and Milstein (Köhler and Milstein,Nature, 256:495-497 [1975]), as well as the trioma technique, the humanB-cell hybridoma technique (See e.g., Kozbor et al., Immunol. Today,4:72 [1983]), and the EBV-hybridoma technique to produce humanmonoclonal antibodies (Cole et al., in Monoclonal Antibodies and CancerTherapy, Alan R. Liss, Inc., pp. 77-96 [1985]).

According to the invention, techniques described for the production ofsingle chain antibodies (U.S. Pat. No. 4,946,778; herein incorporated byreference) can be adapted to produce specific single chain antibodies asdesired. An additional embodiment of the invention utilizes thetechniques known in the art for the construction of Fab expressionlibraries (Huse et al., Science, 246:1275-1281 [1989]) to allow rapidand easy identification of monoclonal Fab fragments with the desiredspecificity.

Antibody fragments that contain the idiotype (antigen binding region) ofthe antibody molecule can be generated by known techniques. For example,such fragments include but are not limited to: the F(ab′)2 fragment thatcan be produced by pepsin digestion of an antibody molecule; the Fab′fragments that can be generated by reducing the disulfide bridges of anF(ab′)2 fragment, and the Fab fragments that can be generated bytreating an antibody molecule with papain and a reducing agent.

Genes encoding antigen binding proteins can be isolated by methods knownin the art. In the production of antibodies, screening for the desiredantibody can be accomplished by techniques known in the art (e.g.,radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), “sandwich”immunoassays, immunoradiometric assays, gel diffusion precipitinreactions, immunodiffusion assays, in situ immunoassays (using colloidalgold, enzyme or radioisotope labels, for example), Western Blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays, etc.), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc.) etc.

As used herein, the term “immunoglobulin” or “antibody” refer toproteins that bind a specific antigen. Immunoglobulins include, but arenot limited to, polyclonal, monoclonal, chimeric, and humanizedantibodies, Fab fragments, F(ab′)₂ fragments, and includesimmunoglobulins of the following classes: IgG, IgA, IgM, IgD, IbE, andsecreted immunoglobulins (slg) Immunoglobulins generally comprise twoidentical heavy chains and two light chains. However, the terms“antibody” and “immunoglobulin” also encompass single chain antibodiesand two chain antibodies.

The term “epitope” as used herein refers to that portion of an antigenthat makes contact with a particular immunoglobulin. When a protein orfragment of a protein is used to immunize a host animal, numerousregions of the protein may induce the production of antibodies whichbind specifically to a given region or three-dimensional structure onthe protein; these regions or structures are referred to as “antigenicdeterminants”. An antigenic determinant may compete with the intactantigen (i.e., the “immunogen” used to elicit the immune response) forbinding to an antibody.

The terms “specific binding” or “specifically binding” when used inreference to the interaction of an antibody and a protein or peptidemeans that the interaction is dependent upon the presence of aparticular structure (i.e., the antigenic determinant or epitope) on theprotein; in other words the antibody is recognizing and binding to aspecific protein structure rather than to proteins in general. Forexample, if an antibody is specific for epitope “A,” the presence of aprotein containing epitope A (or free, unlabelled A) in a reactioncontaining labeled “A” and the antibody will reduce the amount oflabeled A bound to the antibody. As used herein, the terms “non-specificbinding” and “background binding” when used in reference to theinteraction of an antibody and a protein or peptide refer to aninteraction that is not dependent on the presence of a particularstructure (i.e., the antibody is binding to proteins in general ratherthat a particular structure such as an epitope).

As used herein, the term “modulate” refers to the activity of a compound(e.g., benzodiazepine compound) to affect (e.g., to promote or retard)an aspect of cellular function, including, but not limited to, cellgrowth, proliferation, apoptosis, and the like.

As used herein, the term “competes for binding” is used in reference toa first molecule (e.g., a first benzodiazepine derivative) with anactivity that binds to the same substrate (e.g., the oligomycinsensitivity conferring protein in mitochondrial ATP synthase) as does asecond molecule (e.g., a second benzodiazepine derivative or othermolecule that binds to the oligomycin sensitivity conferring protein inmitochondrial ATP synthase, etc.). The efficiency (e.g., kinetics orthermodynamics) of binding by the first molecule may be the same as, orgreater than, or less than, the efficiency of the substrate binding tothe second molecule. For example, the equilibrium binding constant(K_(D)) for binding to the substrate may be different for the twomolecules.

As used herein, the term “instructions for administering said compoundto a subject,” and grammatical equivalents thereof, includesinstructions for using the compositions contained in a kit for thetreatment of conditions characterized by the dysregulation of apoptoticprocesses in a cell or tissue (e.g., providing dosing, route ofadministration, decision trees for treating physicians for correlatingpatient-specific characteristics with therapeutic courses of action).The term also specifically refers to instructions for using thecompositions contained in the kit to treat autoimmune disorders (e.g.,systemic lupus erythematosus, rheumatoid arthritis, graft-versus-hostdisease, myasthenia gravis, Sjögren's syndrome, etc.), chronicinflammatory conditions (e.g., psoriasis, asthma and Crohn's disease),hyperproliferative disorders (e.g., tumors, B cell lymphomas, T celllymphomas, etc.), viral infections (e.g., herpes virus, papilloma virus,HIV), and other conditions such as osteoarthritis and atherosclerosis,and the like.

The term “test compound” refers to any chemical entity, pharmaceutical,drug, and the like, that can be used to treat or prevent a disease,illness, sickness, or disorder of bodily function, or otherwise alterthe physiological or cellular status of a sample (e.g., the level ofdysregulation of apoptosis in a cell or tissue). Test compounds compriseboth known and potential therapeutic compounds. A test compound can bedetermined to be therapeutic by using the screening methods of thepresent invention. A “known therapeutic compound” refers to atherapeutic compound that has been shown (e.g., through animal trials orprior experience with administration to humans) to be effective in suchtreatment or prevention. In preferred embodiments, “test compounds” areagents that modulate apoptosis in cells.

As used herein, the term “third party” refers to any entity engaged inselling, warehousing, distributing, or offering for sale a test compoundcontemplated for administered with a compound for treating conditionscharacterized by the dysregulation of apoptotic processes.

GENERAL DESCRIPTION OF THE INVENTION

As a class of drugs, benzodiazepine compounds have been widely studiedand reported to be effective medicaments for treating a number ofdisease. For example, U.S. Pat. Nos. 4,076,823, 4,110,337, 4,495,101,4,751,223 and 5,776,946, each incorporated herein by reference in itsentirety, report that certain benzodiazepine compounds are effective asanalgesic and anti-inflammatory agents. Similarly, U.S. Pat. Nos.5,324,726 and 5,597,915, each incorporated by reference in its entirety,report that certain benzodiazepine compounds are antagonists ofcholecystokinin and gastrin and thus might be useful to treat certaingastrointestinal disorders.

Other benzodiazepine compounds have been studied as inhibitors of humanneutrophil elastase in the treating of human neutrophilelastase-mediated conditions such as myocardial ischemia, septic shocksyndrome, among others (See e.g., U.S. Pat. No. 5,861,380 incorporatedherein by reference in its entirety). U.S. Pat. No. 5,041,438,incorporated herein by reference in its entirety, reports that certainbenzodiazepine compounds are useful as anti-retroviral agents.

Despite the attention benzodiazepine compounds have drawn, it willbecome apparent from the description below, that the present inventionprovides novel benzodiazepine compounds and related compounds andmethods of using the novel compounds, as well as known compounds, fortreating a variety of diseases.

Benzodiazepine compounds are known to bind to benzodiazepine receptorsin the central nervous system (CNS) and thus have been used to treatvarious CNS disorders including anxiety and epilepsy. Peripheralbenzodiazepine receptors have also been identified, which receptors mayincidentally also be present in the CNS. The present inventiondemonstrates that benzodiazepines and related compounds havepro-apoptotic and cytotoxic properties useful in the treatment oftransformed cells grown in tissue culture. The route of action of thesecompounds is not through the previously identified benzodiazepinereceptors.

Experiments conducted during the development of the present inventionhave identified novel biological targets for benzodiazepine compoundsand related compounds (some of which are related by their ability tobind cellular target molecules rather than their homology to the overallchemical structure of benzodiazepine compounds). In particular, thepresent invention provides compounds that interact, directly orindirectly, with particular mitochondrial proteins to elicit the desiredbiological effects.

Thus, in some embodiments, the present invention provides a number ofnovel compounds and previously known compounds directed against novelcellular targets to achieve desired biological results. In otherembodiments, the present invention provides methods for using suchcompounds to regulate biological processes. The present invention alsoprovides drug-screening methods to identify and optimize compounds. Thepresent invention further provides diagnostic markers for identifyingdiseases and conditions, for monitoring treatment regimens, and/or foridentifying optimal therapeutic courses of action. These and otherresearch and therapeutic utilities are described below.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel chemical compounds, methods fortheir discovery, and their therapeutic use. In particular, the presentinvention provides benzodiazepine derivatives and related compounds andmethods of using benzodiazepine derivatives and related compounds astherapeutic agents to treat a number of conditions associated with thefaulty regulation of the processes of programmed cell death,autoimmunity, inflammation, and hyperproliferation, and the like.

Exemplary compositions and methods of the present invention aredescribed in more detail in the following sections: I. Modulators ofCell Death; II. Modulators of Cell Growth and Proliferation; III.Expression Analysis of Treated Cells; IV. Exemplary Compounds; V.Pharmaceutical compositions, formulations, and exemplary administrationroutes and dosing considerations; VI. Drug screens; and VII. TherapeuticApplications.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of organic chemistry, pharmacology,molecular biology (including recombinant techniques), cell biology,biochemistry, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature, such as,“Molecular cloning: a laboratory manual” Second Edition (Sambrook etal., 1989); “Oligonucleotide synthesis” (M. J. Gait, ed., 1984); “Animalcell culture” (R. I. Freshney, ed., 1987); the series “Methods inenzymology” (Academic Press, Inc.); “Handbook of experimentalimmunology” (D. M. Weir & C. C. Blackwell, eds.); “Gene transfer vectorsfor mammalian cells” (J. M. Miller & M. P. Calos, eds., 1987); “Currentprotocols in molecular biology” (F. M. Ausubel et al., eds., 1987, andperiodic updates); “PCR: the polymerase chain reaction” (Mullis et al.,eds., 1994); and “Current protocols in immunology” (J. E. Coligan etal., eds., 1991), each of which is herein incorporated by reference inits entirety.

I. Modulators of Cell Death

In preferred embodiments, the present invention regulates apoptosisthrough the exposure of cells to compounds. The effect of compounds canbe measured by detecting any number of cellular changes. Cell death maybe assayed as described herein and in the art. In preferred embodiments,cell lines are maintained under appropriate cell culturing conditions(e.g., gas (CO₂), temperature and media) for an appropriate period oftime to attain exponential proliferation without density dependentconstraints. Cell number and or viability are measured using standardtechniques, such as trypan blue exclusion/hemo-cytometry, or MTT dyeconversion assay. Alternatively, the cell may be analyzed for theexpression of genes or gene products associated with aberrations inapoptosis or necrosis.

In preferred embodiments, exposing the present invention to a cellinduces apoptosis. In some embodiments, the present invention causes aninitial increase in cellular ROS levels (e.g., O₂ ⁻). In furtherembodiments, exposure of the compounds of the present invention to acell causes an increase in cellular O₂ ⁻ levels. In still furtherembodiments, the increase in cellular O₂ ⁻ levels resulting from thecompounds of the present invention is detectable with a redox-sensitiveagent that reacts specifically with O₂ ⁻ (e.g., dihyroethedium (DHE)).

In other embodiments, increased cellular O₂ ⁻ levels resulting fromcompounds of the present invention diminish after a period of time(e.g., 10 minutes). In other embodiments, increased cellular O₂ ⁻ levelsresulting from the compounds of the present invention diminish after aperiod of time and increase again at a later time (e.g., 10 hours). Infurther embodiments, increased cellular O₂ ⁻ levels resulting from thecompounds of the present invention diminish at 1 hour and increase againafter 4 hours. In preferred embodiments, an early increase in cellularO₂ ⁻ levels, followed by a diminishing in cellular O₂ ⁻ levels, followedby another increase in cellular O₂ ⁻ levels resulting from the compoundsof the present invention is due to different cellular processes (e.g.,bimodal cellular mechanisms).

In some embodiments, the present invention causes a collapse of a cell'smitochondrial ΔΨ_(m). In preferred embodiments, a collapse of a cell'smitochondrial ΔΨ_(m)resulting from the present invention is detectablewith a mitochondria-selective potentiometric probe (e.g., DiOC₆). Infurther embodiments, a collapse of a cell's mitochondrial ΔΨ_(m)resulting from the present invention occurs after an initial increase incellular O₂ ⁻ levels.

In some embodiments, the present invention enables caspace activation.In other embodiments, the present invention causes the release ofcytochrome c from mitochondria. In further embodiments, the presentinvention alters cystolic cytochrome c levels. In still otherembodiments, altered cystolic cytochrome c levels resulting from thepresent invention are detectable with immunoblotting cytosolicfractions. In preferred embodiments, diminished cystolic cytochrome clevels resulting from the present invention are detectable after aperiod of time (e.g., 10 hours). In further preferred embodiments,diminished cystolic cytochrome c levels resulting from the presentinvention are detectable after 5 hours.

In other embodiments, the present invention causes the opening of themitochondrial PT pore. In preferred embodiments, the cellular release ofcytochrome c resulting from the present invention is consistent with acollapse of mitochondrial ΔΨ_(m). In still further preferredembodiments, the present invention causes an increase in cellular O₂ ⁻levels after a mitochondrial ΔΨ_(m) collapse and a release of cytochromec. In further preferred embodiments, a rise in cellular O₂ ⁻ levels iscaused by a mitochondrial ΔΨ_(m) collapse and release of cytochrome cresulting from the present invention.

In other embodiments, the present invention causes cellular caspaseactivation. In preferred embodiments, caspase activation resulting fromthe present invention is measurable with a pan-caspase sensitivefluorescent substrate (e.g., FAM-VAD-fmk). In still further embodiments,caspase activation resulting from the present invention tracks with acollapse of mitochondrial ΔΨ_(m). In other embodiments, the presentinvention causes an appearance of hypodiploid DNA. In preferredembodiments, an appearance of hypodiploid DNA resulting from the presentinvention is slightly delayed with respect to caspase activation.

In some embodiments, the molecular target for the present invention isfound within mitochondria. In further embodiments, the molecular targetof the present invention involves the mitochondrial ATPase. The primarysources of cellular ROS include redox enzymes and the mitochondrialrespiratory chain (hereinafter MRC). In preferred embodiments,cytochrome c oxidase (complex IV of the MRC) inhibitors (e.g., NaN₃)preclude a present invention dependent increase in cellular ROS levels.In other preferred embodiments, the ubiquinol-cytochrome c reductasecomponent of MRC complex III inhibitors (e.g., FK506) preclude a presentinvention dependent increase in ROS levels.

In some embodiments, an increase in cellular ROS levels due to thecompounds of the present invention result from the binding of thecompounds of the present invention to a target within mitochondria. Inpreferred embodiments, the compounds of the present invention oxidizes2′,7′-dichlorodihydrofluorescin (hereinafter DCF) diacetate to DCF. DCFis a redox-active species capable of generating ROS. In furtherembodiments, the rate of DCF production resulting from the presentinvention increases after a lag period.

Antimycin A generates O₂ ⁻ by inhibiting ubiquinol-cytochrome creductase. In preferred embodiments, the present invention increases therate of ROS production in an equivalent manner to antimycin A. Infurther embodiments, the present invention increases the rate of ROSproduction in an equivalent manner to antimycin A under aerobicconditions supporting state 3 respiration. In further embodiments, thecompounds of the present invention do not directly target the MPT pore.In additional embodiments, the compounds of the present invention do notgenerate substantial ROS in the subcellular S15 fraction (e.g., cytosol;microsomes). In even further embodiments, the compounds of the presentinvention do not stimulate ROS if mitochondria are in state 4respiration.

MRC complexes I-III are the primary sources of ROS within mitochondria.In preferred embodiments, the primary source of an increase in cellularROS levels resulting from the dependent invention emanates from thesecomplexes as a result of inhibiting the mitochondrial F₁F₀-ATPase.Indeed, in still further embodiments, the present invention inhibitsmitochondrial ATPase activity of bovine sub-mitochondrial particles(hereinafter SMPs). In particularly preferred embodiments, the compoundsof the present invention bind to the OSCP component of the mitochondrialF₁F₀-ATPase.

In some embodiments, the compounds of the present invention have thestructure:

or its enantiomer, wherein, R₁ is aliphatic or aryl; R₂ is aliphatic,aryl, —NH₂, —HC(═O)—R₅, or a moiety that participates in hydrogen bondformation, wherein R₅ is aryl, heterocyclic, —R₆—NH—C(═O)—R₇ or—R₆—C(═O)—NH—R₇, wherein R₆ is an aliphatic linker of 1-6 carbons and R₇is aliphatic, aryl, or heterocyclic; and each of R₃ and R₄ isindependently hydrogen, hydroxy, alkoxy, halo, amino,lower-alkyl-substituted-amino, acylamino, hydroxyamino, an aliphaticgroup having 1-8 carbons and 1-20 hydrogens, aryl, or heteroaryl; or apharmaceutically acceptable salt, prodrug or derivative thereof In somepreferred embodiments, where R3 is a hydroxyl group, one or moreadditional positions on the ring containing R3 includes a chemical group(e.g., an alkyl chain) that protects the hydroxyl group from metabolismin vivo.

In certain embodiments, the compounds of the present invention may havea hydroxyl group at the C′4 position and an aromatic ring. In preferredembodiments, compounds of the present invention cause an increase incellular ROS levels as a result of a hydroxyl group at the C′4 positionand an aromatic ring. In further embodiments, the potency of the presentinvention in cell based assays correlates with ATPase inhibitionexperiments using SMPs. Indeed, in preferred embodiments, the presentinvention significantly inhibits mitochondrial ATPase activity incomparison to cytotoxic (80 μM) concentrations of generalbenzodiazepines and PBR ligands (e.g., PK11195 and 4-chlorodiazepam)that do not significantly inhibit mitochondrial ATPase activity. Assuch, in preferred embodiments, the molecular target of the presentinvention is the mitochondrial ATPase.

Oligomycin is a macrolide natural product that binds to themitochondrial F₁F₀-ATPase, induces a state 3 to 4 transition, and as aresult, generates ROS (e.g., O₂ ⁻). In preferred embodiments, thepresent invention binds the OSCP component of the mitochondrialF₁F₀-ATPase. In certain embodiments, screening assays of the presentinvention permit detection of binding partners of the OSCP. OSCP is anintrinsically fluorescent protein. In certain embodiments, titrating asolution of test compounds of the present invention into an E. Colisample overexpressed with OSCP results in quenching of the intrinsicOSCP fluorescence. In other embodiments, fluorescent or radioactive testcompounds can be used in direct binding assays. In other embodiments,competition binding experiments can be conducted. In this type of assay,test compounds are assessed for their ability to compete with Bz-423 forbinding to the OSCP. In some embodiments, the compounds of the presentinvention cause a reduced increase in cellular ROS levels and reducedapoptosis in cells through regulation of the OSCP gene (e.g., alteringexpression of the OSCP gene). In further embodiments, the presentinvention functions by altering the molecular motions of the ATPasemotor.

II. Modulators of Cellular Proliferation and Cell Growth

In some embodiments, the compounds and methods of the present inventioncauses descreased cellular proliferation. In other embodiments, thecompounds and methods of the present invention causes decreased cellularproliferation and apoptosis. For example, cell culture cytotoxicityassays conducted during the development of the present inventiondemonstrated that the compounds and methods of the present inventionprevents cell growth after an extended period in culture (e.g., 3 days).

III. Expression Analysis of Treated Cells

In some embodiments, induced cell death is not dependent upon newprotein synthesis. Treatment of cells with cyclohexamide inhibits newprotein synthesis. In some embodiments, cells treated with cyclohexamideand the compounds of the present invention enter apoptosis.

During the development of the present invention, an expression profilewas generated to identify those genes that are differentially expressedin treated and untreated cells. This profile provides a gene expressionfingerprint of cells induced by the compounds of the present invention.This fingerprint identifies genes that are upregulated and downregulatedin response to the compounds of the present invention and identifiessuch genes are diagnostic markers for drug screening and for monitoringtherapeutic effects of the compounds. The genes also provide targets forregulation to mimic the effects of the compounds of the presentinvention. Data from an expression analysis for genes up-regulated inthe presence of Bz-423 is presented in FIG. 4A. Data from an expressionanalysis for genes down-regulated in the presence of Bz-423 is presentedin FIG. 4B. Data from an expression analysis for genes up-regulated inthe presence of Bz-OMe is presented in FIG. 4C. Data from an expressionanalysis for genes down-regulated in the presence of Bz-OMe is presentedin FIG. 4D.

For example, an analysis of the expression profile provides ornithinedecarboxylase antizyme 1 (OAZ1) as a novel therapeutic agent. OAZ1 is animportant regulatory protein that controls the synthesis and transportinto cells of polyamines, including putrescine, spermidine and spermineThe synthesis of poylamines in cells involves several enzymatic steps,however ornithine decarboxylase is the enzyme that principally regulatesthis process. By inhibiting the polyamine transporter located in theplasma membrane and by targeting ornithine decarboxylase for proteolyticdegradation, OAZ1 reduces polyamine levels in cells. Polyamines areessential for the survival and growth of cells. Abnormal accumulation ofpolyamines contributes to tumor induction, cancer growth and metastasisInhibitors of polyamine biosynthesis, and specifically one moleculeidentified as difluoromethylornithine (DFMO), are in clinical trials toconfirm their anticarcinogenic and therapeutic potential. In preferredembodiments of the present invention, OAZ1 is induced to a level 16-foldabove the level of control cells in cells treated with the compounds ofthe present invention. Any method, direct or indirect, for inducing OAZ1levels is contemplated by the present invention (e.g., treatment withcompounds of the present invention, gene therapy, etc.).

OAZ1 is an important regulator of polyamine metabolism and functions todecrease polyamine levels by acting as an inhibitor of ornithinedecarboxylase (ODC), a mitochondrial enzyme that controls therate-limiting step of polyamine biosynthesis. After inhibition withantizyme, ODC is targeted for proteosomal degredation. Polyamines areintimately involved in cellular stability and required for cellproliferation. Inhibiting polyamine synthesis suppresses proliferation.As such, in still further embodiments, ODC expression or activity isdecreased (e.g., using siRNA, antisense oligonucleotides, gene therapy,known or later identified inhibitors, the compounds of the presentinvention, etc.) to elicit the desired biological effect.

Antizyme 1 expression is regulated transcriptionally and at thepost-transcriptional level. Post-transcriptional regulation plays aparticularly important role in the regulation of this gene product andoccurs by a unique translational frameshift that depends on eitherpolymanes (through a negative-feedback loop) or agmatine, anothermetabolite of arginine. ODC activity leves may be obtained by quanifyingthe conversion of ornithine to putrescine using ³H-ornithine. In someembodiments, treating cells with the compounds of the present inventionsignificantly reduces ODC activity in a dose-dependant fashion. In stillfurther embodiments, a reduction in ODC activity is paralleled by adecrease in ODC protein levels measured under similar conditions. Cellspre-incubated with MnTBAP decrease ROS levels. In some embodiments,cells pre-incubated with MnTBAP that are exposed to the compounds of thepresent invention display reversed inhibition of ODC.

In preferred embodiments, cells treated with high levels (e.g., >10 μM)of the compounds of the present invention generate sufficient amounts ofROS that are not detoxified by cellular anti-oxidants, and result inapoptosis within a short time period (e.g., 18 h). In preferredembodiments, cells treated with lower levels (e.g., <10 μM) of thecompounds of the present invention induce a reduced ROS response that isinsufficient to trigger apoptosis, but is capable of inhibiting ODC orotherwise blocking cellular proliferation. In other embodiments, aderivative of the compounds of the present invention in which thephenolic hydroxyl is replaced by Cl or OCH₃ is minimally cytotoxic,generates a small ROS response in cells, binds less tightly to the OSCP,and inhibits ODC activity. In still other embodiments, cells treatedwith a derivative of the compounds of the present invention in which thephenolic hydroxyl is replaced by Cl experience reduced proliferation toa similar extent as to the unmodified compounds. As such, in preferredembodiments, the antiproliferative effects are obtained using chemicalderivatives of the compounds of the present invention that blockproliferation without inducing apoptosis.

In response to antigenic or mitogenic stimulation, lymphocytes secreteprotein mediators, one of which is named migration inhibitory factor(MIF) for its ability to prevent the migration of macrophages in vitro.MIF may be an anti-tumor agent. In addition, the ability of MIF toprevent the migration of macrophages may be exploited for treatingwounds. MIF may alter the immune response to different antigens. MIFlinks chemical and immunological detoxification systems. MIF was inducedapproximately 10-fold by Bz-423. Thus, the present inventioncontemplates the use of MIF as a target of the compounds of the presentinvention.

Prolifin is induced at high levels in cell treated with the presentinvention. Profilin binds to actin monomers and interacts with severalproteins and phosphoinositides, linking signaling pathways to thecytoskeleton. Profilin can sequester actin monomers, increase exchangeof ATP for ADP on actin, and increase the rate of actin filamentturnover. A comparison between several different tumorigenic cancer celllines with nontumorigenic lines show consistently lower profilin 1levels in tumor cells. Transfection of profilin 1 cDNA into CAL51 breastcancer cells raised the profilin 1 level, had a prominent effect on cellgrowth, and suppressed tumorigenicity of the overexpressing cell clonesin nude mice. Therefore, induction of profilin 1 (e.g., by the compoundsof the present invention or otherwise) may suppress the tumorigenesis ofcancer cells.

Interferon regulatory factor 4 (IRF-4) is induced at higher than normallevels in cells treated with the compounds of the present invention.IRF-4 is a lymphoid/myeloid-restricted member of the IRF transcriptionfactor family that plays an essential role in the homeostasis andfunction of mature lymphocytes. IRF-4 expression is regulated in restingprimary T cells and is transiently induced at the mRNA and proteinlevels after activation by stimuli such as TCR cross-linking ortreatment with phorbol ester and calcium ionophore (PMA/ionomycin).Stable expression of IRF-4 in Jurkat cells leads to a strong enhancementin the synthesis of interleukin (IL)-2, IL-4, IL-10, and IL-13. IRF-4represents one of the lymphoid-specific components that control theability of T lymphocytes to produce a distinctive array of cytokines. InAbelson-transformed pro-B cell lines, enforced expression of IRF-4 issufficient to induce germline Igk transcription. The action of thecompounds of the present invention to induce IRF-4 may account for itsaffects on autoimmune disease in B and T cell dominant processes as wellas for its ability to influence the survival of neoplastic B cellclones.

In preferred embodiments, cell death-regulatory protein GRIM19 isinduced at higher than normal levels in cells treated with the compoundsof the present invention. The importance of the interferon (IFN) pathwayin cell growth suppression is known. Studies have shown that acombination of IFN and all-trans retinoic acid inhibits cell growth invitro and in vivo more potently than either agent alone. The specificgenes that play a role in IFN/RA-induced cell death were identified byan antisense knockout approach, and called GRIM genes. GRIM19 is a novelcell death-associated gene that is not included in any of the knowndeath gene categories. This gene encodes a 144-aa protein that localizesto the nucleus. Overexpression of GRIM19 enhances caspase-9 activity andapoptotic cell death in response to IFN/RA treatment. GRIM19 is locatedin the 19p13.2 region of the human chromosome essential for prostatetumor suppression, signifying that the protein may be a novel tumorsuppressor. The induction of GRIM19 by the compounds of the presentinvention may result in anti-tumor effects.

IV. Exemplary Compounds

Exemplary compounds of the present invention are provided below.

or its enantiomer, wherein, R₁ is aliphatic or aryl; R₂ is aliphatic,aryl, —NH₂, —NHC(═O)—R₅; or a moiety that participates in hydrogenbonding, wherein R₅ is aryl, heterocyclic, —R₆—NH—C(═O)—R₂ or—R₆—C(═O)—NH—R₇, wherein R₆ is an aliphatic linker of 1-6 carbons and R₇is aliphatic, aryl, or heterocyclic, each of R₃ and R₄ is independentlya hydroxy, alkoxy, halo, amino, lower-alkyl-substituted-amino,acetylamino, hydroxyamino, an aliphatic group having 1-8 carbons and1-20 hydrogens, aryl, or heterocyclic; or a pharmaceutically acceptablesalt, prodrug or derivative thereof.

In the above structures, R₁ is a hydrocarbyl group of 1-20 carbons and1-20 hydrogens. Preferably, R₁ has 1-15 carbons, and more preferably,has 1-12 carbons. Preferably, R₁ has 1-12 hydrogens, and morepreferably, 1-10 hydrogens. Thus R₁ can be an aliphatic group or an arylgroup.

The term “aliphatic” represents the groups commonly known as alkyl,alkenyl, alkynyl, alicyclic. The term “aryl” as used herein represents asingle aromatic ring such as a phenyl ring, or two or more aromaticrings that are connected to each other (e.g., bisphenyl) or fusedtogether (e.g., naphthalene or anthracene). The aryl group can beoptionally substituted with a lower aliphatic group (e.g., C₁-C₄ alkyl,alkenyl, alkynyl, or C₃-C₆ alicyclic). Additionally, the aliphatic andaryl groups can be further substituted by one or more functional groupssuch as —NH₂, —NHCOCH₃, —OH, lower alkoxy (C₁-C₄), halo (—F, —Cl, —Br,or —I). It is preferable that R₁ is primarily a nonpolar moiety.

In the above structures, R₂ can be aliphatic, aryl, —NH₂, —NHC(═O)—R₅,or a moiety that participates in hydrogen bonding, wherein R₅, is aryl,heterocyclic, R₆—NH—C(═O)—R₇ or —R₆—C(═O)—NH—R₇, wherein R₆ is analiphatic linker of 1-6 carbons and R₇ is an aliphatic, aryl, orheterocyclic. The terms “aliphatic” and “aryl” are as defined above.

The term “a moiety that participates in hydrogen bonding” as used hereinrepresents a group that can accept or donate a proton to form a hydrogenbond thereby.

Some specific non-limiting examples of moieties that participate inhydrogen bonding include a fluoro, oxygen-containing andnitrogen-containing groups that are well-known in the art. Some examplesof oxygen-containing groups that participate in hydrogen bondinginclude: hydroxy, lower alkoxy, lower carbonyl, lower carboxyl, lowerethers and phenolic groups. The qualifier “lower” as used herein refersto lower aliphatic groups (C₁-C₄) to which the respectiveoxygen-containing functional group is attached.

Thus, for example, the term “lower carbonyl” refers to inter alia,formaldehyde, acetaldehyde.

Some nonlimiting examples of nitrogen-containing groups that participatein hydrogen bond formation include amino and amido groups. Additionally,groups containing both an oxygen and a nitrogen atom can alsoparticipate in hydrogen bond formation. Examples of such groups includenitro, N-hydroxy and nitrous groups.

It is also possible that the hydrogen-bond acceptor in the presentinvention can be the Π electrons of an aromatic ring. However, thehydrogen bond participants of this invention do not include those groupscontaining metal atoms such as boron. Further the hydrogen bonds formedwithin the scope of practicing this invention do not include thoseformed between two hydrogens, known as “dihydrogen bonds.” (See, R. H.Crabtree, Science, 282:2000-2001 [1998], for further description of suchdihydrogen bonds).

The term “heterocyclic” represents, for example, a 3-6 membered aromaticor nonaromatic ring containing one or more heteroatoms. The heteroatomscan be the same or different from each other. Preferably, at least oneof the heteroatom's is nitrogen. Other heteroatoms that can be presenton the heterocyclic ring include oxygen and sulfur.

Aromatic and nonaromatic heterocyclic rings are well-known in the art.Some nonlimiting examples of aromatic heterocyclic rings includepyridine, pyrimidine, indole, purine, quinoline and isoquinoline.Nonlimiting examples of nonaromatic heterocyclic compounds includepiperidine, piperazine, morpholine, pyrrolidine and pyrazolidine.Examples of oxygen containing heterocyclic rings include, but notlimited to furan, oxirane, 2H-pyran, 4H-pyran, 2H-chromene, andbenzofuran. Examples of sulfur-containing heterocyclic rings include,but are not limited to, thiophene, benzothiophene, and parathiazine.

Examples of nitrogen containing rings include, but not limited to,pyrrole, pyrrolidine, pyrazole, pyrazolidine, imidazole, imidazoline,imidazolidine, pyridine, piperidine, pyrazine, piperazine, pyrimidine,indole, purine, benzimidazole, quinoline, isoquinoline, triazole, andtriazine.

Examples of heterocyclic rings containing two different heteroatomsinclude, but are not limited to, phenothiazine, morpholine,parathiazine, oxazine, oxazole, thiazine, and thiazole.

The heterocyclic ring is optionally further substituted with one or moregroups selected from aliphatic, nitro, acetyl (i.e., —C(═O)—CH₃), oraryl groups.

Each of R₃ and R₄ can be independently a hydroxy, alkoxy, halo, amino,or substituted amino (such as lower-alkyl-substituted-amino, oracetylamino or hydroxyamino), or an aliphatic group having 1-8 carbonsand 1-20 hydrogens. When each of R₃ and R₄ is an aliphatic group, it canbe further substituted with one or more functional groups such as ahydroxy, alkoxy, halo, amino or substituted amino groups as describedabove. The terms “aliphatic” is defined above. Alternatively, each of R₃and R₄ can be hydrogen.

It is well-known that many 1,4-benzodiazepines exist as optical isomersdue to the chirality introduced into the heterocyclic ring at tile C₃position. The optical isomers are sometimes described as L- or D-isomersin the literature. Alternatively, the isomers are also referred to as R-and S-enantiomorphs. For the sake of simplicity, these isomers arereferred to as enantiomorphs or enantiomers. The 1,4-benzodiazepinecompounds described herein include their enantiomeric forms as well asracemic mixtures. Thus, the usage “benzodiazepine or its enantiomers”herein refers to the benzodiazepine as described or depicted, includingall its enantiomorphs as well as their racemic mixture.

From the above description, it is apparent that many specific examplesare represented by the generic formulas presented above. Thus, in oneexample, R₁ is aliphatic, R₂ is aliphatic, whereas in another example,R₁ is aryl and R₂ is a moiety that participates in hydrogen bondformation. Alternatively, R₁ can be aliphatic, and R₂ can be an—NHC(═O)—R₅, or a moiety that participates in hydrogen bonding, whereinR₅ is aryl, heterocyclic, —R₆—NH—C(═O)—R₇ or —R₆—C(═O)—NH—R₇, wherein R₆is an aliphatic linker of 1-6 carbons and R₇ is an aliphatic, aryl, orheterocyclic. A wide variety of sub combinations arising from selectinga particular group at each substituent position are possible and allsuch combinations are within the scope of this invention.

Further, it should be understood that the numerical ranges giventhroughout this disclosure should be construed as a flexible range thatcontemplates any possible subrange within that range. For example, thedescription of a group having the range of 1-10 carbons would alsocontemplate a group possessing a subrange of, for example, 1-3, 1-5,1-8, or 2-3, 2-5, 2-8, 3-4, 3-5, 3-7, 3-9, 3-10, etc., carbons. Thus,the range 1-10 should be understood to represent the outer boundaries ofthe range within which many possible subranges are clearly contemplated.Additional examples contemplating ranges in other contexts can be foundthroughout this disclosure wherein such ranges include analogoussubranges within. Some specific examples of the benzodiazepine compoundsof this invention include:

wherein R₂ is

and dimethylphenyl (all isomers) and ditrifluoromethyl (all isomers).

The following compounds are also contemplated:

This invention also provides the compound Bz-423.

Bz-423 differs from benzodiazepines in clinical use by the presence of ahydrophobic substituent at C-3. This substitution renders binding to theperipheral benzodiazepine receptor (“PBR”) weak (K_(d) ca. 1 μM) andprevents binding to the central benzodiazepine receptor so that Bz-423is not a sedative.

In some embodiments R2 is any chemical group that permits the compoundto bind to OSCP. In some such embodiments, R2 comprises a hydrophobicaromatic group. In preferred embodiments R2 comprises a hydrophobicaromatic group larger than benzene (e.g., a benzene ring withnon-hydrogen substituents, a moiety having two or more aromatic rings, amoiety with 7 or more carbon atoms, etc.).

Additional specific benzodiazepine derivative examples of the presentinvention include the following:

R1 is H or hydroxyEach of R2 through R6 may be the same or different and is selected fromhydrogen, a hydroxy, an alkoxy, a halo, an amino, a lower-alkyl-asubstituted-amino, an acetylamino, a hydroxyamino, an aliphatic grouphaving 1-8 carbons and 1-20 hydrogens, a substituted aliphatic group ofsimilar size, a cycloaliphatic group consisting of <10 carbons, asubstituted cycloaliphatic group, an aryl, and a heterocyclic

Each of R1 through R10 may be the same or different and is selected fromhydrogen, a hydroxy, an alkoxy, a halo, an amino, a lower-alkyl-asubstituted-amino, an acetylamino, a hydroxyamino, an aliphatic grouphaving 1-8 carbons and 1-20 hydrogens, a substituted aliphatic group ofsimilar size, a cycloaliphatic group consisting of <10 carbons, asubstituted cycloaliphatic group, an aryl, and a heterocyclic

Each of R1 through R11 may be the same or different and is selected fromhydrogen, a hydroxy, an alkoxy, a halo, an amino, a lower-alkyl-asubstituted-amino, an acetylamino, a hydroxyamino, an aliphatic grouphaving 1-8 carbons and 1-20 hydrogens, a substituted aliphatic group ofsimilar size, a cycloaliphatic group consisting of <10 carbons, asubstituted cycloaliphatic group, an aryl, and a heterocyclic

Each of R1 through R10 may be the same or different and is selected fromhydrogen, a hydroxy, an alkoxy, a halo, an amino, a lower-alkyl-asubstituted-amino, an acetylamino, a hydroxyamino, an aliphatic grouphaving 1-8 carbons and 1-20 hydrogens, a substituted aliphatic group ofsimilar size, a cycloaliphatic group consisting of <10 carbons, asubstituted cycloaliphatic group, an aryl, and a heterocyclic

Each of R1 through R10 may be the same or different and is selected fromhydrogen, a hydroxy, an alkoxy, a halo, an amino, a lower-alkyl-asubstituted-amino, an acetylamino, a hydroxyamino, an aliphatic grouphaving 1-8 carbons and 1-20 hydrogens, a substituted aliphatic group ofsimilar size, a cycloaliphatic group consisting of <10 carbons, asubstituted cycloaliphatic group, an aryl, and a heterocyclic

Each of R1 through R6 may be the same or different and is selected fromhydrogen, a hydroxy, an alkoxy, a halo, an amino, a lower-alkyl-asubstituted-amino, an acetylamino, a hydroxyamino, an aliphatic grouphaving 1-8 carbons and 1-20 hydrogens, a substituted aliphatic group ofsimilar size, a cycloaliphatic group consisting of <10 carbons, asubstituted cycloaliphatic group, an aryl, and a heterocyclic

wherein R₁ is selected from napthalalanine; phenol; 1-Napthalenol;2-Napthalenol;

and quinolines.A composition comprising the following formula:

wherein R₁ is selected from:

The stereochemistry of all derivatives embodied in the present inventionis R, S, or racemic.

In summary, a large number of benzodiazepine compounds and relatedcompounds are presented herein. Any one or more of these compounds canbe used to treat a variety of dysregulatory disorders related tocellular death as described elsewhere herein. The above-describedcompounds can also be used in drug screening assays and other diagnosticmethods.

V. Pharmaceutical Compositions, Formulations, and ExemplaryAdministration Routes and Dosing Considerations

Exemplary embodiments of various contemplated medicaments andpharmaceutical compositions are provided below.

A. Preparing Medicaments

The compounds of the present invention are useful in the preparation ofmedicaments to treat a variety of conditions associated withdysregulation of cell death, aberrant cell growth andhyperproliferation.

In addition, the compounds are also useful for preparing medicaments fortreating other disorders wherein the effectiveness of the compounds areknown or predicted. Such disorders include, but are not limited to,neurological (e.g., epilepsy) or neuromuscular disorders. The methodsand techniques for preparing medicaments of a compound are well-known inthe art. Exemplary pharmaceutical formulations and routes of deliveryare described below.

One of skill in the art will appreciate that any one or more of thecompounds described herein, including the many specific embodiments, areprepared by applying standard pharmaceutical manufacturing procedures.Such medicaments can be delivered to the subject by using deliverymethods that are well-known in the pharmaceutical arts.

B. Exemplary Pharmaceutical Compositions and Formulation

In some embodiments of the present invention, the compositions areadministered alone, while in some other embodiments, the compositionsare preferably present in a pharmaceutical formulation comprising atleast one active ingredient/agent (e.g., benzodiazepine derivative), asdefined above, together with a solid support or alternatively, togetherwith one or more pharmaceutically acceptable carriers and optionallyother therapeutic agents. Each carrier must be “acceptable” in the sensethat it is compatible with the other ingredients of the formulation andnot injurious to the subject.

Contemplated formulations include those suitable oral, rectal, nasal,topical (including transdermal, buccal and sublingual), vaginal,parenteral (including subcutaneous, intramuscular, intravenous andintradermal) and pulmonary administration. In some embodiments,formulations are conveniently presented in unit dosage form and areprepared by any method known in the art of pharmacy. Such methodsinclude the step of bringing into association the active ingredient withthe carrier which constitutes one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association (e.g., mixing) the active ingredient withliquid carriers or finely divided solid carriers or both, and then ifnecessary shaping the product.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tablets,wherein each preferably contains a predetermined amount of the activeingredient; as a powder or granules; as a solution or suspension in anaqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion ora water-in-oil liquid emulsion. In other embodiments, the activeingredient is presented as a bolus, electuary, or paste, etc.

In some embodiments, tablets comprise at least one active ingredient andoptionally one or more accessory agents/carriers are made by compressingor molding the respective agents. In preferred embodiments, compressedtablets are prepared by compressing in a suitable machine the activeingredient in a free-flowing form such as a powder or granules,optionally mixed with a binder (e.g., povidone, gelatin,hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative,disintegrant (e.g., sodium starch glycolate, cross-linked povidone,cross-linked sodium carboxymethyl cellulose)surface-active or dispersingagent. Molded tablets are made by molding in a suitable machine amixture of the powdered compound (e.g., active ingredient) moistenedwith an inert liquid diluent. Tablets may optionally be coated or scoredand may be formulated so as to provide slow or controlled release of theactive ingredient therein using, for example, hydroxypropylmethylcellulose in varying proportions to provide the desired release profile.Tablets may optionally be provided with an enteric coating, to providerelease in parts of the gut other than the stomach.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavored basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouthwashes comprising the active ingredient in asuitable liquid carrier.

Pharmaceutical compositions for topical administration according to thepresent invention are optionally formulated as ointments, creams,suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosolsor oils. In alternatively embodiments, topical formulations comprisepatches or dressings such as a bandage or adhesive plasters impregnatedwith active ingredient(s), and optionally one or more excipients ordiluents. In preferred embodiments, the topical formulations include acompound(s) that enhances absorption or penetration of the activeagent(s) through the skin or other affected areas. Examples of suchdermal penetration enhancers include dimethylsulfoxide (DMSO) andrelated analogues.

If desired, the aqueous phase of a cream base includes, for example, atleast about 30% w/w of a polyhydric alcohol, i.e., an alcohol having twoor more hydroxyl groups such as propylene glycol, butane-1,3-diol,mannitol, sorbitol, glycerol and polyethylene glycol and mixturesthereof.

In some embodiments, oily phase emulsions of this invention areconstituted from known ingredients in an known manner. This phasetypically comprises an lone emulsifier (otherwise known as an emulgent),it is also desirable in some embodiments for this phase to furthercomprises a mixture of at least one emulsifier with a fat or an oil orwith both a fat and an oil.

Preferably, a hydrophilic emulsifier is included together with alipophilic emulsifier so as to act as a stabilizer. It some embodimentsit is also preferable to include both an oil and a fat. Together, theemulsifier(s) with or without stabilizer(s) make up the so-calledemulsifying wax, and the wax together with the oil and/or fat make upthe so-called emulsifying ointment base which forms the oily dispersedphase of the cream formulations.

Emulgents and emulsion stabilizers suitable for use in the formulationof the present invention include Tween 60, Span 80, cetostearyl alcohol,myristyl alcohol, glyceryl monostearate and sodium lauryl sulfate.

The choice of suitable oils or fats for the formulation is based onachieving the desired properties (e.g., cosmetic properties), since thesolubility of the active compound/agent in most oils likely to be usedin pharmaceutical emulsion formulations is very low. Thus creams shouldpreferably be a non-greasy, non-staining and washable products withsuitable consistency to avoid leakage from tubes or other containers.Straight or branched chain, mono- or dibasic alkyl esters such asdi-isoadipate, isocetyl stearate, propylene glycol diester of coconutfatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate,butyl stearate, 2-ethylhexyl palmitate or a blend of branched chainesters known as Crodamol CAP may be used, the last three being preferredesters. These may be used alone or in combination depending on theproperties required. Alternatively, high melting point lipids such aswhite soft paraffin and/or liquid paraffin or other mineral oils can beused.

Formulations suitable for topical administration to the eye also includeeye drops wherein the active ingredient is dissolved or suspended in asuitable carrier, especially an aqueous solvent for the agent.

Formulations for rectal administration may be presented as a suppositorywith suitable base comprising, for example, cocoa butter or asalicylate.

Formulations suitable for vaginal administration may be presented aspessaries, creams, gels, pastes, foams or spray formulations containingin addition to the agent, such carriers as are known in the art to beappropriate.

Formulations suitable for nasal administration, wherein the carrier is asolid, include coarse powders having a particle size, for example, inthe range of about 20 to about 500 microns which are administered in themanner in which snuff is taken, i.e., by rapid inhalation (e.g., forced)through the nasal passage from a container of the powder held close upto the nose. Other suitable formulations wherein the carrier is a liquidfor administration include, but are not limited to, nasal sprays, drops,or aerosols by nebulizer, an include aqueous or oily solutions of theagents.

Formulations suitable for parenteral administration include aqueous andnon-aqueous isotonic sterile injection solutions which may containantioxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents, and liposomes or other microparticulatesystems which are designed to target the compound to blood components orone or more organs. In some embodiments, the formulations arepresented/formulated in unit-dose or multi-dose sealed containers, forexample, ampoules and vials, and may be stored in a freeze-dried(lyophilized) condition requiring only the addition of the sterileliquid carrier, for example water for injections, immediately prior touse. Extemporaneous injection solutions and suspensions may be preparedfrom sterile powders, granules and tablets of the kind previouslydescribed.

Preferred unit dosage formulations are those containing a daily dose orunit, daily subdose, as herein above-recited, or an appropriate fractionthereof, of an agent.

It should be understood that in addition to the ingredients particularlymentioned above, the formulations of this invention may include otheragents conventional in the art having regard to the type of formulationin question, for example, those suitable for oral administration mayinclude such further agents as sweeteners, thickeners and flavoringagents. It also is intended that the agents, compositions and methods ofthis invention be combined with other suitable compositions andtherapies. Still other formulations optionally include food additives(suitable sweeteners, flavorings, colorings, etc.), phytonutrients(e.g., flax seed oil), minerals (e.g., Ca, Fe, K, etc.), vitamins, andother acceptable compositions (e.g., conjugated linoelic acid),extenders, and stabilizers, etc.

C. Exemplary Administration Routes and Dosing Considerations

Various delivery systems are known and can be used to administer atherapeutic agents (e.g., benzodiazepine derivatives) of the presentinvention, e.g., encapsulation in liposomes, microparticles,microcapsules, receptor-mediated endocytosis, and the like. Methods ofdelivery include, but are not limited to, intra-arterial,intra-muscular, intravenous, intranasal, and oral routes. In specificembodiments, it may be desirable to administer the pharmaceuticalcompositions of the invention locally to the area in need of treatment;this may be achieved by, for example, and not by way of limitation,local infusion during surgery, injection, or by means of a catheter.

The agents identified herein as effective for their intended purpose canbe administered to subjects or individuals susceptible to or at risk ofdeveloping pathological growth of target cells and condition correlatedwith this. When the agent is administered to a subject such as a mouse,a rat or a human patient, the agent can be added to a pharmaceuticallyacceptable carrier and systemically or topically administered to thesubject. To determine patients that can be beneficially treated, atissue sample is removed from the patient and the cells are assayed forsensitivity to the agent.

Therapeutic amounts are empirically determined and vary with thepathology being treated, the subject being treated and the efficacy andtoxicity of the agent. When delivered to an animal, the method is usefulto further confirm efficacy of the agent. One example of an animal modelis MLR/MpJ-lpr/lpr (“MLR-lpr”) (available from Jackson Laboratories, BalHarbor, Me.). MLR-lpr mice develop systemic autoimmune disease.Alternatively, other animal models can be developed by inducing tumorgrowth, for example, by subcutaneously inoculating nude mice with about10⁵ to about 10⁹ hyperproliferative, cancer or target cells as definedherein. When the tumor is established, the compounds described hereinare administered, for example, by subcutaneous injection around thetumor. Tumor measurements to determine reduction of tumor size are madein two dimensions using venier calipers twice a week. Other animalmodels may also be employed as appropriate. Such animal models for theabove-described diseases and conditions are well-known in the art.

In some embodiments, in vivo administration is effected in one dose,continuously or intermittently throughout the course of treatment.Methods of determining the most effective means and dosage ofadministration are well known to those of skill in the art and vary withthe composition used for therapy, the purpose of the therapy, the targetcell being treated, and the subject being treated. Single or multipleadministrations are carried out with the dose level and pattern beingselected by the treating physician.

Suitable dosage formulations and methods of administering the agents arereadily determined by those of skill in the art. Preferably, thecompounds are administered at about 0.01 mg/kg to about 200 mg/kg, morepreferably at about 0.1 mg/kg to about 100 mg/kg, even more preferablyat about 0.5 mg/kg to about 50 mg/kg. When the compounds describedherein are co-administered with another agent (e.g., as sensitizingagents), the effective amount may be less than when the agent is usedalone.

The pharmaceutical compositions can be administered orally,intranasally, parenterally or by inhalation therapy, and may take theform of tablets, lozenges, granules, capsules, pills, ampoules,suppositories or aerosol form. They may also take the form ofsuspensions, solutions and emulsions of the active ingredient in aqueousor nonaqueous diluents, syrups, granulates or powders. In addition to anagent of the present invention, the pharmaceutical compositions can alsocontain other pharmaceutically active compounds or a plurality ofcompounds of the invention.

More particularly, an agent of the present invention also referred toherein as the active ingredient, may be administered for therapy by anysuitable route including, but not limited to, oral, rectal, nasal,topical (including, but not limited to, transdermal, aerosol, buccal andsublingual), vaginal, parental (including, but not limited to,subcutaneous, intramuscular, intravenous and intradermal) and pulmonary.It is also appreciated that the preferred route varies with thecondition and age of the recipient, and the disease being treated.

Ideally, the agent should be administered to achieve peak concentrationsof the active compound at sites of disease. This may be achieved, forexample, by the intravenous injection of the agent, optionally insaline, or orally administered, for example, as a tablet, capsule orsyrup containing the active ingredient.

Desirable blood levels of the agent may be maintained by a continuousinfusion to provide a therapeutic amount of the active ingredient withindisease tissue. The use of operative combinations is contemplated toprovide therapeutic combinations requiring a lower total dosage of eachcomponent antiviral agent than may be required when each individualtherapeutic compound or drug is used alone, thereby reducing adverseeffects.

D. Exemplary Co-Administration Routes and Dosing Considerations

The present invention also includes methods involving co-administrationof the compounds described herein with one or more additional activeagents. Indeed, it is a further aspect of this invention to providemethods for enhancing prior art therapies and/or pharmaceuticalcompositions by co-administering a compound of this invention. Inco-administration procedures, the agents may be administeredconcurrently or sequentially. In one embodiment, the compounds describedherein are administered prior to the other active agent(s). Thepharmaceutical formulations and modes of administration may be any ofthose described above. In addition, the two or more co-administeredchemical agents, biological agents or radiation may each be administeredusing different modes or different formulations.

The agent or agents to be co-administered depends on the type ofcondition being treated. For example, when the condition being treatedis cancer, the additional agent can be a chemotherapeutic agent orradiation. When the condition being treated is an autoimmune disorder,the additional agent can be an immunosuppressant or an anti-inflammatoryagent. When the condition being treated is chronic inflammation, theadditional agent can be an anti-inflammatory agent. The additionalagents to be co-administered, such as anticancer, immunosuppressant,anti-inflammatory, and can be any of the well-known agents in the art,including, but not limited to, those that are currently in clinical use.The determination of appropriate type and dosage of radiation treatmentis also within the skill in the art or can be determined with relativeease.

Treatment of the various conditions associated with abnormal apoptosisis generally limited by the following two major factors: (1) thedevelopment of drug resistance and (2) the toxicity of known therapeuticagents. In certain cancers, for example, resistance to chemicals andradiation therapy has been shown to be associated with inhibition ofapoptosis. Some therapeutic agents have deleterious side effects,including non-specific lymphotoxicity, renal and bone marrow toxicity.

The methods described herein address both these problems. Drugresistance, where increasing dosages are required to achieve therapeuticbenefit, is overcome by co-administering the compounds described hereinwith the known agent. The compounds described herein appear to sensitizetarget cells to known agents (and vice versa) and, accordingly, less ofthese agents are needed to achieve a therapeutic benefit.

The sensitizing function of the claimed compounds also addresses theproblems associated with toxic effects of known therapeutics. Ininstances where the known agent is toxic, it is desirable to limit thedosages administered in all cases, and particularly in those cases weredrug resistance has increased the requisite dosage. When the claimedcompounds are co-administered with the known agent, they reduce thedosage required which, in turn, reduces the deleterious effects.Further, because the claimed compounds are themselves both effective andnon-toxic in large doses, co-administration of proportionally more ofthese compounds than known toxic therapeutics will achieve the desiredeffects while minimizing toxic effects.

VI. Drug Screens

In preferred embodiments of the present invention, the compounds of thepresent invention, and other potentially useful compounds, are screenedfor their binding affinity to the oligomycin sensitivity conferringprotein (OSCP) portion of the mitochondrial ATP synthase complex. Inparticularly preferred embodiments, compounds are selected for use inthe methods of the present invention by measuring their biding affinityto recombinant OSCP protein. A number of suitable screens for measuringthe binding affinity of drugs and other small molecules to receptors areknown in the art. In some embodiments, binding affinity screens areconducted in in vitro systems. In other embodiments, these screens areconducted in in vivo or ex vivo systems. While in some embodimentsquantifying the intracellular level of ATP following administration ofthe compounds of the present invention provides an indication of theefficacy of the methods, preferred embodiments of the present inventiondo not require intracellular ATP level quantification.

Additional embodiments are directed to measuring levels (e.g.,intracellular) of superoxide in cells and/or tissues to measure theeffectiveness of particular contemplated methods and compounds of thepresent invention. In this regard, those skilled in the art willappreciate and be able to provide a number of assays and methods usefulfor measuring superoxide levels in cells and/or tissues.

In some embodiments, structure-based virtual screening methodologies arecontemplated for predicting the binding affinity of compounds of thepresent invention with OSCP.

Any suitable assay that allows for a measurement of the rate of bindingor the affinity of a benzodiazepine or other compound to the OSCP may beutilized. Examples include, but are not limited to, competition bindingusing Bz-423, surface plasma resonace (SPR) andradio-immunopreciptiation assays (Lowman et al., J. Biol. Chem.266:10982 [1991]). Surface Plasmon Resonance techniques involve asurface coated with a thin film of a conductive metal, such as gold,silver, chrome or aluminum, in which electromagnetic waves, calledSurface Plasmons, can be induced by a beam of light incident on themetal glass interface at a specific angle called the Surface PlasmonResonance angle. Modulation of the refractive index of the interfacialregion between the solution and the metal surface following binding ofthe captured macromolecules causes a change in the SPR angle which caneither be measured directly or which causes the amount of lightreflected from the underside of the metal surface to change. Suchchanges can be directly related to the mass and other optical propertiesof the molecules binding to the SPR device surface. Several biosensorsystems based on such principles have been disclosed (See e.g., WO90/05305). There are also several commercially available SPR biosensors(e.g., BiaCore, Uppsala, Sweden).

In some embodiments, copmpounds are screened in cell culture or in vivo(e.g., non-human or human mammals) for their ability to modulatemitochondrial ATP synthase activity. Any suitable assay may be utilized,including, but not limited to, cell proliferation assays (Commerciallyavailable from, e.g., Promega, Madison, WI and Stratagene, La Jolla,Calif.) and cell based dimerization assays. (See e.g., Fuh et al.,Science, 256:1677 [1992]; Colosi et al., J. Biol. Chem., 268:12617[1993]). Additional assay formats that find use with the presentinvention include, but are not limited to, assays for measuring cellularATP levels, and cellular superoxide levels.

The present invention also provides methods of modifying andderivatizing the compositions of the present invention to increasedesirable properties (e.g., binding affinity, activity, and the like),or to minimize undesirable properties (e.g., nonspecific reactivity,toxicity, and the like). The principles of chemical derivatization arewell understood. In some embodiments, iterative design and chemicalsynthesis approaches are used to produce a library of derivatized childcompounds from a parent compound. In other embodiments, rational designmethods are used to predict and model in silico ligand-receptorinteractions prior to confirming results by routine experimentation.

VII. Therapeutic Application

In particularly preferred embodiments, the compositions (e.g.,benzodiazepine derivatives) of the present invention provide therapeuticbenefits to patients suffering from any one or more of a number ofconditions (e.g., diseases characterized by dysregulation of necrosisand/or apoptosis processes in a cell or tissue, disease characterized byaberrant cell growth and/or hyperproliferation, etc.) by modulating(e.g., inhibiting or promoting) the activity of the mitochondrial ATPsynthase (as referred to as mitochondrial F₀F₁ ATPase) complexes inaffected cells or tissues. In further preferred embodiments, thecompositions of the present invention are used to treatautoimmune/chronic inflammatory conditions (e.g., psoriasis).

In particularly preferred embodiments, the compositions of the presentinvention inhibit the activity of mitochondrial ATP synthase complex bybinding to a specific subunit of this multi-subunit protein complex.While the present invention is not limited to any particular mechanism,nor to any understanding of the action of the agents being administered,in some embodiments, the compositions of the present invention bind tothe oligomycin sensitivity conferring protein (OSCP) portion of themitochondrial ATP synthase complex. Likewise, it is further contemplatedthat when the compositions of the present invention bind to the OSCP theinitial affect is overall inhibition of the mitochondrial ATP synthasecomplex, and that the downstream consequence of binding is a change inATP level and the production of reactive oxygen species (e.g., O₂—). Instill other preferred embodiments, while the present invention is notlimited to any particular mechanism, nor to any understanding of theaction of the agents being administered, it is contemplated that thegeneration of free radicals ultimately results in cell killing. In yetother embodiments, while the present invention is not limited to anyparticular mechanism, nor to any understanding of the action of theagents being administered, it is contemplated that the inhibitingmitochondrial ATP synthase complex using the compositions and methods ofthe present invention provides therapeutically useful inhibition of cellproliferation.

Accordingly, preferred methods embodied in the present invention,provide therapeutic benefits to patients by providing compounds of thepresent invention that modulate (e g., inhibiting or promoting) theactivity of the mitochondrial ATP synthase complexes in affected cellsor tissues via binding to the oligomycin sensitivity conferring protein(OSCP) portion of the mitochondrial ATP synthase complex. Importantly,by itself the OSCP has no biological activity.

Thus, in one broad sense, preferred embodiments of the present inventionare directed to the discovery that many diseases characterized bydysregulation of necrosis and/or apoptosis processes in a cell ortissue, or diseases characterized by aberrant cell growth and/orhyperproliferation, etc., can be treated by modulating the activity ofthe mitochondrial ATP synthase complex including, but not limited to, bybinding to the oligomycin sensitivity conferring protein (OSCP)component thereof. The present invention is not intended to be limited,however, to the practice of the compositions and methods explicitlydescribed herein. Indeed, those skilled in the art will appreciate thata number of additional compounds not specifically recited herein (e.g.,non-benzodiazepine derivatives) are suitable for use in the methodsdisclosed herein of modulating the activity of mitochondrial ATPsynthase.

The present invention thus specifically contemplates that any number ofsuitable compounds presently known in the art, or developed later, canoptionally find use in the methods of the present invention. Forexample, compounds including, but not limited to, oligomycin, ossamycin,cytovaricin, apoptolidin, bafilomyxcin, resveratrol, piceatannol, anddicyclohexylcarbodiimide (DCCD), and the like, find use in the methodsof the present invention. The present invention is not intended,however, to be limited to the methods or compounds specified above. Inone embodiment, that compounds potentially useful in the methods of thepresent invention may be selected from those suitable as described inthe scientific literature. (See e.g., K. B. Wallace and A. A. Starkov,Annu. Rev. Pharmacol. Toxicol., 40:353-388 [2000]; A.R. Solomon et al.,Proc. Nat. Acad. Sci. U.S.A., 97(26):14766-14771 [2000]).

In some embodiments, compounds potentially useful in methods of thepresent invention are screened against the National Cancer Institute's(NCI-60) cancer cell lines for efficacy. (See e.g., A. Monks et al., J.Natl. Cancer Inst., 83:757-766 [1991]; and K. D. Paull et al., J. Natl.Cancer Inst., 81:1088-1092 [1989]). Additional screens suitable screens(e.g., autoimmunity disease models, etc.) are within the skill in theart.

In one aspect, derivatives (e.g., pharmaceutically acceptable salts,analogs, stereoisomers, and the like) of the exemplary compounds orother suitable compounds are also contemplated as being useful in themethods of the present invention.

Those skilled in the art of preparing pharmaceutical compounds andformulations will appreciate that when selecting optional compounds foruse in the methods disclosed herein, that suitability considerationsinclude, but are not limited to, the toxicity, safety, efficacy,availability, and cost of the particular compounds.

EXAMPLES

The following examples are provided to demonstrate and furtherillustrate certain preferred embodiments of the present invention andare not to be construed as limiting the scope thereof.

Example 1 Preparation of Compounds

The benzodiazepine compounds are prepared using either solid-phase orsoluble-phase combinatorial synthetic methods as well as on anindividual basis from well-established techniques. See, for example,Boojamra, C. G. et al. (1996); Bunin, B. A., et al. (1994); Stevens, S.Y. et al., (1996); Gordon, E. M., et al., (1994); and U.S. Pat. Nos.4,110,337 and 4,076,823, which are all incorporated by reference herein.For illustration, the following general methodologies are provided.

Preparation of 1,4-benzodiazepine-2-one Compounds

Improved solid-phase synthetic methods for the preparation of a varietyof 1,4-benzodiazepine-2-one derivatives with very high overall yieldshave been reported in the literature. (See e.g., Bunin and Ellman, J.Am. Chem. Soc., 114:10997-10998 [1992]). Using these improved methods,the 1,4-benzodiazepine-2-ones is constructed on a solid support fromthree separate components: 2-aminobenzophenones, α-amino acids, and(optionally) alkylating agents.

Preferred 2-aminobenzophenones include the substituted2-aminobenzophenones, for example, the halo-, hydroxy-, andhalo-hydroxy-substituted 2-aminobenzophenones, such as4-halo-4′-hydroxy-2-aminobenzophenones. A preferred substituted2-aminobenzophenone is 4-chloro-4′-hydroxy-2-aminobenzophenone.Preferred α-amino acids include the 20 common naturally occurringα-amino acids as well as α-amino acid mimicking structures, such ashomophenylalanine, homotyrosine, and thyroxine.

Alkylating agents include both activated and inactivated electrophiles,of which a wide variety are well known in the art. Preferred alkylatingagents include the activated electrophiles p-bromobenzyl bromide andt-butyl-bromoacetate.

In the first step of such a synthesis, the 2-aminobenzophenonederivative is attached to a solid support, such as a polystyrene solidsupport, through either a hydroxy or carboxylic acid functional groupusing well known methods and employing an acid-cleavable linker, such asthe commercially available [4-(hydroxymethyl)phenoxy]acetic acid, toyield the supported 2-aminobenzophenone. (See e.g., Sheppard andWilliams, Intl. J. Peptide Protein Res., 20:451-454 [1982]). The 2-aminogroup of the aminobenzophenone is preferably protected prior to reactionwith the linking reagent, for example, by reaction with FMOC-Cl(9-fluorenylmethyl chloroformate) to yield the protected amino group2′-NHFMOC.

In the second step, the protected 2-amino group is deprotected (forexample, the —NHFMOC group may be deprotected by treatment withpiperidine in dimethylformamide (DMF)), and the unprotected2-aminobenzophenone is then coupled via an amide linkage to an α-aminoacid (the amino group of which has itself been protected, for example,as an —NHFMOC group) to yield the intermediate. Standard activationmethods used for general solid-phase peptide synthesis are used (such asthe use of carbodiimides and hydroxybentzotriazole or pentafluorophenylactive esters) to facilitate coupling. However, a preferred activationmethod employs treatment of the 2-aminobenzophenone with a methylenechloride solution of the of α-N-FMOC-amino acid fluoride in the presenceof the acid scavenger 4-methyl-2,6-di-tert-butylpyridine yields completecoupling via an amide linkage. This preferred coupling method has beenfound to be effective even for unreactive aminobenzophenone derivatives,yielding essentially complete coupling for derivatives possessing both4-chloro and 3-carboxy deactivating substituents.

In the third step, the protected amino group (which originated with theamino acid) is first deprotected (e.g., -NHFMOC may be converted to —NH₂with piperidine in DMF), and the deprotected Bz-423s reacted with acid,for example, 5% acetic acid in DMF at 60° C., to yield the supported1,4-benzodiazepine derivative. Complete cyclization has been reportedusing this method for a variety of 2-aminobenzophenone derivatives withwidely differing steric and electronic properties.

In an optional fourth step, the 1,4-benzodiazepine derivative isalkylated, by reaction with a suitable alkylating agent and a base, toyield the supported fully derivatized 1,4-benzodiazepine. Standardalkylation methods, for example, an excess of a strong base such as LDA(lithium diisopropylamide) or NaH, is used; however, such methods mayresult in undesired deprotonation of other acidic functionalities andover-alkylation. Preferred bases, which may prevent over-alkylation ofthe benzodiazepine derivatives (for example, those with ester andcarbamate functionalities), are those which are basic enough tocompletely deprotonate the anilide functional group, but not basicenough to deprotonate amide, carbamate or ester functional groups. Anexample of such a base is lithiated 5-(phenylmethyl)-2-oxaxolidinone,which is reacted with the 1,4-benzodiazepine in tetrahydrofuran (THF) at−78° C. Following deprotonation, a suitable alkylating agent, asdescribed above, is added.

In the final step, the fully derivatized 1,4-benzodiazepine is cleavedfrom the solid support. This is achieved (along with concomitant removalof acid-labile protecting groups), for example, by exposure to asuitable acid, such as a mixture of trifluoroacetic acid, water, anddimethylsulfide (85:5:10, by volume). Alternatively, the abovebenzodiazepines is prepared in soluble phase. The synthetic methodologywas outlined by Gordon et al., J. Med. Chem., 37:1386-1401 [1994]) whichis hereby incorporated by reference. Briefly, the methodology comprisestrans-imidating an amino acid resin with appropriately substituted2-aminobenzophenone imines to form resin-bound imines. These imines arecyclized and tethered by procedures similar to those in solid-phasesynthesis described above. The general purity of benzodiazepinesprepared using the above methodology is about 90% or higher.

Preparation of 1,4-benzodiazepine-2,5-diones

A general method for the solid-phase synthesis of1,4-benzodiazepine-2,5-diones has been reported in detail by C. J.Boojamra et al., J. Org. Chem., 62:1240-1256 [1996]). This method isused to prepare the compounds of the present invention.

A Merrifield resin, for example, a (chloromethyl)polystyrene isderivatized by alkylation with 4-hydroxy-2,6-dimethoxybenzaldehydesodium to provide resin-bound aldehyde. An α-amino ester is thenattached to the derivatized support by reductive amination usingNaBH(OAc)₃ in 1% acetic acid in DMF. This reductive amination results inthe formation of a resin-bound secondary amine

The secondary amine is acylated with a wide variety of unprotectedanthranilic acids result in support-bound tertiary amides. Acylation isbest achieved by performing the coupling reaction in the presence of acarbodiimide and the hydrochloride salt of a tertiary amine One goodcoupling agent is 1-ethyl-8-[8-(dimethylamino)propyl]carbodiimidehydrochloride. The reaction is typically performed in the presence ofanhydrous 1-methyl-2-pyrrolidinone. The coupling procedure is typicallyrepeated once more to ensure complete acylation.

Cyclization of the acyl derivative is accomplished throughbase-catalyzed lactamation through the formation of an anilide anionwhich would react with an alkylhalide for simultaneous introduction ofthe substituent at the 1-position on the nitrogen of the heterocyclicring of the benzodiazepine. The lithium salt of acetanilide is a goodbase to catalyze the reaction. Thus, the Bz-423s reacted with lithiumacetanilide in DMF/THF (1:1) for 30 hours followed by reaction withappropriate alkylating agent provides the fully derivatizedsupport-bound benzodiazepine. The compounds are cleaved from the supportin good yield and high purity by using TFA/DMS/H₂O (90:5:5).

Some examples of the α-amino ester starting materials, alkylatingagents, and anthranilic acid derivatives that are used in the presentinvention are listed by Boojamra (1996), supra at 1246. Additionalreagents are readily determined and either are commercially obtained orreadily prepared by one of ordinary skill in the art to arrive at thenovel substituents disclosed in the present invention.

For example, from Boojamra, supra, one realizes that: alkylating agentsprovide the R₁ substituents; α-amino ester starting materials providethe R₂ substituents, and anthranilic acids provide the R₄ substituents.By employing these starting materials that are appropriatelysubstituted, one arrives at the desired 1,4-benzodiazepine-2,5-dione.The R₃ substituent is obtained by appropriately substituting the amineof the α-aminoester starting material. If steric crowding becomes aproblem, the R₃ substituent is attached through conventional methodsafter the 1,4-benzodiazepine-2,5-dione is isolated.

Example 2 Chirality

It should be recognized that many of the benzodiazepines of the presentinvention exist as optical isomers due to chirality wherein thestereocenter is introduced by the α-amino acid and its ester startingmaterials. The above-described general procedure preserves the chiralityof the α-amino acid or ester starting materials. In many cases, suchpreservation of chirality is desirable. However, when the desiredoptical isomer of the α-amino acid or ester starting material isunavailable or expensive, a racemic mixture is produced which isseparated into the corresponding optical isomers and the desiredbenzodiazepine enantiomer is isolated.

For example, in the case of the 2,5-dione compounds, Boojamra, supra,discloses that complete racemization is accomplished by preequilibratingthe hydrochloride salt of the enantiomerically pure α-amino esterstarting material with 0.3 equivalents of i-Pr₂EtN and the resin-boundaldehyde for 6 hours before the addition of NaBH(OAc)₃. The rest of theabove-described synthetic procedure remains the same. Similar steps areemployed, if needed, in the case of the 1,4-benzodiazepine-2-dionecompounds as well. Methods to prepare individual benzodiazepines arewell-known in the art. (See e.g., U.S. Pat. Nos. 3,415,814; 3,384,635;and 3,261,828, which are hereby incorporated by reference). By selectingthe appropriately substituted starting materials in any of theabove-described methods, the benzodiazepines of this invention areprepared with relative ease.

Example 3 Reagents

Bz-423 is synthesized as described above. FK506 is obtained fromFujisawa (Osaka, Japan).N-benzoylcarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD) is obtainedfrom Enzyme Systems (Livermore, Calif.). Dihydroethidium (DHE) and3,3′-dihexyloxacarbocyanine iodide (DiOC₆(3)) are obtained fromMolecular Probes (Eugene, Oreg.). FAM-VAD-fmk is obtained from Intergen(Purchase, NJ). Manganese(III)meso-tetrakis(4-benzoic acid)porphyrin(MnTBAP) is purchased from Alexis Biochemicals (San Diego, Calif.).Benzodiazepines is synthesized as described (See, B.A. Bunin et al.,Proc. Natl. Acad. Sci. U.S.A., 91:4708-4712 [1994]). Other reagents wereobtained from Sigma (St. Louis, Mo.).

Example 4 Animals and Drug Delivery

Female NZB/W mice (Jackson Labs, Bar Harbor, Me.) are randomlydistributed into treatment and control groups. Control mice receivevehicle (50 μL aqueous DMSO) and treatment mice receive Bz-423 dissolvedin vehicle (60 mg/kg) through intraperitoneal injections. Peripheralblood is obtained from the tail veins for the preparation of serum.Samples of the spleen and kidney are preserved in either 10%buffered-formalin or by freezing in OCT. An additional section of spleenfrom each animal is reserved for the preparation of single cellsuspensions.

Example 5 Primary Splenocytes, Cell Lines, and Culture Conditions

Primary splenocytes are obtained from 6 month old mice by mechanicaldisruption of spleens with isotonic lysis of red blood cells. Bcell-rich fractions are prepared by negative selection using magneticcell sorting with CD4, CD8a and CD11b coated microbeads (MiltenyiBiotec, Auburn, Calif.). The Ramos line is purchased from the ATCC(Monassis, Ga.). Cells are maintained in RPMI supplemented with 10%heat-inactivated fetal bovine serum (FBS), penicillin (100 U/ml),streptomycin (100 μg/ml) and L-glutamine (290 μg/ml). Media for primarycells also contains 2-mercaptoethanol (50 μM). All in vivo studies areperformed with 0.5% DMSO and 2% FBS. In vitro experiments are conductedin media containing 2% FBS. Organic compounds are dissolved in mediacontaining 0.5% DMSO.

Example 6 Histology

Formalin-fixed kidney sections were stained with hematoxylin and eosin(H&E) and glomerular immune-complex deposition is detected by directimmunofluorescence using frozen tissue stained with FITC-conjugated goatanti-mouse IgG (Southern Biotechnology, Birmingham, Ala.). Sections areanalyzed in a blinded fashion for nephritis and IgG deposition using a0-4+ scale. The degree of lymphoid hyperplasia is scored on a 0-4+ scaleusing spleen sections stained with H&E. To identify B cells, sectionsare stained with biotinylated-anti-B220 (Pharmingen; 1 μg/mL) followedby streptavidin-Alexa 594 (Molecular Probes; 5 μg/mL). Frozen spleensections are analyzed for TUNEL positive cells using an In situ CellDeath Detection kit (Roche) and are evaluated using a 0-4+ scale.

Example 7 TUNEL Staining

Frozen spleen sections are analyzed using an In situ Cell DeathDetection kit (Roche Molecular Biochemicals, Indianapolis, IN). Sectionsare blindly evaluated and assigned a score (0-4+) on the basis of theamount of TUNEL-positive staining. B cells are identified by stainingwith biotinylated-anti-B220 (Pharmingen, San Diego, Calif.; 1 μg/mL, 1h, 22° C.) followed by streptavidin-Alexa 594 (Molecular Probes, Eugene,Oreg.; 5 μg/mL, 1 h, 22° C.).

Example 8 Flow Cytometric Analysis of Spleen Cells from Treated Animals

Surface markers are detected (15 m, 4° C.) with fluorescent-conjugatedanti-Thy 1.2 (Pharmingen, 1 μg/mL) and/or anti-B220 (Pharmingen, 1μg/mL). To detect outer-membrane phosphatidyl serine, cells areincubated with FITC-conjugated Annexin V and propidium iodide (PI)according to manufacturer protocols (Roche Molecular Biochemicals).Detection of TUNEL-positive cells by flow cytometry uses the APO-BRDUkit (Pharmingen). Superoxide and MPT are assessed by incubation of cellsfor 30 m at 27 degrees C. with 10 μM dihydroethidium and 2 μM3,3′-dihexyloxacarbocyanine iodide (DIOC₆(3)) (Molecular Probes).Prodidium idodie is used to determine viability and DNA content. Samplesare analyzed on a FACSCalibur flow cytometer (Becton Dickinson, SanDiego, Calif.).

Example 9 B Cell Stimulation

Ramos cells are activated with soluble goat Fab₂ anti-human IgM(Southern Biotechnology Associates, 1 μpg/ml) and/or purified anti-humanCD40 (Pharmingen, clone 5C3, 2.5 μg/ml). Mouse B cells are activatedwith affinity purified goat anti-mouse IgM (ICN, Aurora, Ohio; 20 μg/ml)immobilized in culture wells, and/or soluble purified anti-mouse CD40(Pharmingen, clone HM40-3, 2.5 μg/ml). LPS is used at 10 μg/ml. Bz-423is added to cultures immediately after stimuli are applied Inhibitorsare added 30 m prior to Bz-423.

Example 10 Statistical Analysis

Statistical analysis is conducted using the SPSS software package.Statistical significance is assessed using the Mann-Whitney U test andcorrelation between variables is assessed by two-way ANOVA. All p-valuesreported are one-tailed and data are presented as mean±SEM.

Example 11 Detection of Cell death and Hypodiploid DNA

Cell viability is assessed by staining with propidium iodide (PI, 1μg/mL). PI fluorescence is measured using a FACScalibur flow cytometer(Becton Dickinson, San Diego, Calif.). Measurement of hypodiploid DNA isconducted after incubating cells in DNA-labeling solution (50 μg/mL ofPI in PBS containing 0.2% Triton and 10 μg/mL RNAse A) overnight at 4degrees C. The data is analyzed using the CellQuest software excludingaggregates.

Example 12 Detection of O₂ ⁻, ψ_(m), and Caspase Activation

To detect O₂ ⁻, cells are incubated with DHE (10 μM) for 30 min at 37°C. and are analyzed by flow cytometry to measure ethidium fluorescence.Flow analysis of mitochondrial transmembrane potential (ψ_(m)) isconducted by labeling cells with DiOC₆(3) (20 nM) for 15 min at 37degrees C. A positive control for disruption of ψ_(m) is establishedusing carbonyl cyanide m-chlorophenylhydrazone (CCCP, 50 μM). Caspaseactivation assays are performed with FAM-VAD-fluoromethylketone.Processing of the substrate is evaluated by flow cytometry.

Example 13 Subcellular Fractionation and Cytochrome c Detection

Ramos cells (250×10⁶ cells/sample) are treated with Bz-423 (10 μM) orvehicle for 1 to 5 h. Cells are pelleted, re-suspended in buffer (68 mMsucrose, 220 mM mannitol, 10 mM HEPES-NaOH, pH 7.4, 10 mM KCl, 1 mMEDTA, 1 mM EGTA, 10 μl g/mL leupeptin, 10 μg/mL aprotinin, 1 mM PMSF),incubated on ice for 10 min, and homogenized. The homogenate iscentrifuged twice for 5 min at 4° C. (800 g) to pellet nuclei and debrisand for 15 min at 4° C. (16,000 g) to pellet mitochondria. Thesupernatant is concentrated, electrophoresed on 12% SDS-PAGE gels, andtransferred to Hybond ECL membranes (Amersham, Piscataway, N.J.). Afterblocking (PBS containing 5% dried milk and 0.1% Tween), the membranesare probed with an anti-cytochrome c monoclonal antibody (Pharmingen,San Diego, Calif.; 2 μg/mL) followed by an anti-mouse horseradishperoxidase-conjugated secondary with detection by chemiluminescence(Amersham).

Example 14 ROS Production in Isolated Mitochondria

Male Long Evans rats are starved overnight and sacrificed bydecapitation. Liver samples are homogenized in ice cold buffer A (250 mMsucrose, 10 mM Tris, 0.1 mM EGTA, pH 7.4), and nuclei and cellulardebris are pelleted (10 mM, 830g, 4° C.). Mitochondria are collected bycentrifugation (10 min, 15,000g, 4° C.), and the supernatant iscollected as the S15 fraction. The mitochondrial pellet is washed threetimes with buffer B (250 mM sucrose, 10 mM Tris, pH 7.4), andre-suspended in buffer B at 20-30 mg/mL. Mitochondria are diluted (0.5mg/mL) in buffer C (200 mM sucrose, 10 mM Tris, pH 7.4, 1 mM KH₂PO₄, 10μM EGTA, 2.5 μM rotenone, 5 mM succinate) containing2′,7′-dichlorodihydrofluorescin diacetate (DCFH-DA, 1 μM). For state 3measurements, ADP (2 mM) is included in the buffer, and prior to theaddition of Bz-423, mitochondria are allowed to charge for 2 min. Toinduce state 4, oligomycin (10 μM) is added to buffer C. The oxidationof DCFH to 2′,7′-dichlorofluorescein (DCF) is monitored at 37° C. with aspectrofluorimeter (λ_(ex): 503 nm; λ_(em): 522 nm). To detect effectson O₂ ⁻ and delta ψ_(m), mitochondria are incubated for 15 min at 37° C.in buffer C with vehicle, Bz-423, or CCCP containing DHE (5 μM) orDIOC₆(3) (20 nM), and aliquots are removed for analysis by fluorescencemicroscopy.

Example 15 Flow Cytometric Analysis of Splenocytes

Splenocytes are prepared by mechanical disruption and red blood cellsremoved by isotonic lysis. Cells are stained at 4° C. withfluorescent-conjugated anti-Thy 1.2 (Pharmingen; 1 μg/mL) and/oranti-B220 (Pharmingen; 1 μg/mL) for 15 min. To detect outer-membranephosphatidyl serine, cells are incubated with FITC-conjugated Annexin Vand PI (Roche Molecular Biochemicals, Indianapolis, Ind.; 1 μg/mL).

Example 16 In vivo Determination of ROS

Spleens are removed from 4-mo old NZB/W mice treated with Bz-423 orvehicle and frozen in OCT. ROS production is measured usingmanganese(II)3,3,9-diaminobenzidine as described in E. D. Kerver et al.(See, E. D. Kerver et al., Histochem. J., 29:229-237 [1997).

Example 17 IgG Titers, BUN, and Proteinuria

Anti-DNA and IgG titers are determined by ELISA as described in P. C.Swanson et al. (See, P. C. Swanson et al., Biochemistry, 35:1624-1633[1996]). Serum BUN is measured by the University of Michigan Hospital'sclinical laboratory. Proteinuria is monitored using ChemStrip 6(Boehringer Mannheim).

Example 18 Benzodiazepine Studies

Benzodiazepine studies on animals are described in U.S. Patent No.:20010016583, published Aug. 23, 2001, herein incorporated by referencein its entirety.

Example 19 Mediators of Bz-423 Induced Apoptosis

To characterize the death mechanism engaged by Bz-423, intracellularROS, ΔΨ_(m), cytochrome c release, caspase activation, and DNAfragmentation were measured over time (the results presented are for Bcells but do characterize the response in many different cell types).The first event detected after exposure to Bz-423 is an increase in thefraction of cells that stain with dihyroethedium (DHE), aredox-sensitive agent that reacts specifically with O₂ ⁻.

Levels of O₂ ⁻ diminished after an early maximum at 1 hour and thenincreased again after 4 hours of continued treatment. This bimodalpattern pointed to a cellular mechanism limiting O₂ ⁻ and suggested thatthe “early” and “late” O₂ ⁻ maxima resulted from different processes.

Collapse of ΔΨ_(m) was detected using DiOC₆(3), a mitochondria-selectivepotentiometric probe. The gradient change began after the early O₂ ⁻response and was observed in >90% of cells by 5 hours.

Cytochrome c release from mitochondria, a key step enabling caspaseactivation, was studied by immunoblotting cytosolic fractions. Levels ofcytosolic cytochrome c above amounts in cells treated with vehicle weredetected by 5 hours. This release was coincident with the disruption ofΔΨ_(m), and together, these results were consistent with opening of thePT pore. Indeed, the late increase in O₂ ⁻ tracked with the ΔΨ_(m)collapse and the release of cytochrome c, suggesting that the secondaryrise in O₂ ⁻ resulted from these processes.

Caspase activation was measured by processing of the pan-caspasesensitive fluorescent substrate FAM-VAD-fmk. Caspase activation trackedwith ΔΨ_(m), whereas the appearance of hypodiploid DNA was slightlydelayed with respect to caspase activation. Collectively, these resultsindicated that Bz-423 induces a mitochondrial-dependent apoptoticpathway.

Example 20 Bz-423 Directly Targets Mitochondria

Since the early O₂ ⁻ preceded other cellular events, it was possiblethat this ROS had a regulatory role. In non-phagocytic cells, redoxenzymes, along with the MRC, are the primary sources of ROS. Inhibitorsof these systems were assayed for an ability to regulate Bz-423-inducedO₂ ⁻ in order to determine the basis for this response. Of thesereagents, only NaN₃, which acts primarily on cytochrome c oxidase(complex IV of the mitochondrial respiratory chain, MRC), and micromolaramounts of FK506, which block the formation of O₂ ⁻ by theubiquinol-cytochrome c reductase component of MRC complex III, modulatedBz-423. These findings suggested that mitochondria are the source ofBz-423 -induced O₂ ⁻ and that a component of the MRC is involved in theresponse. Although the inhibition by FK506 may result from binding toeither calcineurin or FK506-binding proteins, natural products that bindtightly to these proteins (rapamycin and cyclosporin A, respectively)did not diminish the Bz-423 O₂ ⁻ response.

O₂ ⁻ production by Bz-423 may result from binding to a protein withinmitochondria or a target in another compartment that signalsmitochondria to generate ROS. To distinguish between these alternatives,isolated rat liver mitochondria were assayed for ROS production bymonitoring the oxidation of 2′,7′-dichlorodihydrofluorescin diacetate toof 2′,7′-dichlorofluorescin in the presence and absence of Bz-423. Inthis assay, the rate of DCF production increased after a lag periodduring which endogenous reducing equivalents were consumed and theacetate moieties on the probe were hydrolyzed to yield2′,7′-dichlorodihydrofluorescin, the redox-active species. Under aerobicconditions supporting state 3 respiration, both antimycin A, whichgenerates O₂ ⁻ by inhibiting ubiquinol-cytochrome c reductase, andBz-423 increased the rate of ROS production nearly two-fold after theinduction phase, based on comparing the slopes of each curve to control.Swelling was not observed, demonstrating that Bz-423 does not directlytarget the MPT pore. Neither Bz-423 nor antimycin A generatedsubstantial ROS in the subcellular S15 fraction (cytosol andmicrosomes), and Bz-423 does not stimulate ROS if mitochondria are instate 4, even though antimycin A is active under these conditions.Together, these experiments demonstrate that mitochondria contain amolecular target for Bz-423, and state 3 respiration is required for theO₂ ⁻ response.

Example 21 Bz-423-Induced ROS Comes from Mitochondria

MRC complexes I and III are the primary sources of ROS withinmitochondria. Evidence presented above suggests that Bz-423 -induced ROScomes from mitochondria. To test this hypothesis, MRC function wasknocked out the resulting cells were examined for

ROS in response to Bz-423. Complexes I-IV in the MRC are partiallyencoded by mitochondrial DNA (mtDNA). Culturing cells over extendedperiods of time in the presence of ethidium bromide removed mtDNA,suggesting that mtDNA encoded proteins are not produced and electrontransport along the MRC does not occur (cells devoid of mtDNA andassociated proteins are often termed ρ⁰ cells). Because ethidium bromideis toxic to Ramos cells, these experiments were conducted with Namalwa Bcells, another mature B cell line. Treating Namalwa ρ⁰ cells with Bz-423did not result in an ROS response, as was observed in both Ramos andNamalwa ρ⁺ cells.

Since the early ROS is critical to Bz-423 induced apoptosis, resultsdetected with the Namalwa ρ⁰ cells would seemingly predict that thesecells would be protected from the toxic effects of Bz-423. However,after 6 hours, the MPT was triggered and Namalwa ρ⁰ cells underwentapoptosis in response to Bz-423. In ρ⁺ cells, proton pumping by the MRCmaintained the mitochondrial gradient ΔΨ_(m). Since a functional MRC isnot present in ρ⁰ cells, ΔΨ_(m) is supported by complex V (theF₁F₀-ATPase) functioning as an ATPase (deletion of subunits 6 and b inρ⁰ cells abolishes the synthase activity of this enzyme). In this case,inhibition of complex V ATPase would cause collapse of the gradient andsubsequent cell death.

Example 22 Bz-423 Targets the Mitochondrial F₁F₀-ATPase

Oligomycin, a macrolide natural product that binds to the mitochondrialF₁F₀-ATPase, induces a state 3 to 4 transition and generates O₂ ⁻ likeBz-423. Based on these similarities, it is possible that the F₁F₀-ATPaseis also the molecular target for Bz-423. To test this hypothesis, theeffect of Bz-423 on ATPase activity in sub-mitochondrial particles(SMPs) was examined. Indeed, Bz-423 inhibited the mitochondrial ATPaseactivity of bovine SMPs with an ED 50 ca. 5 μM.

>40 derivatives of Bz-423 were developed to determine the elements onthis novel agent required for biological activity. Assessing thesecompounds in whole cell apoptosis assays revealed that a hydroxyl groupat the C′4 position and an aromatic ring roughly the size of the napthylmoiety were useful. The potency of these analogues in cell based assayscorrelated with the ED₅₀ values in ATPase inhibition experiments usingSMPs. These observations indicated that the mitochondrial ATPase is themolecular target of Bz-423. At concentrations where these derivativesare cytotoxic (80 μM), other benzodiazepines and PBR ligands (e.g.,PK11195 and 4-chlorodiazepam) do not significantly inhibit mitochondrialATPase activity, suggesting that the molecular target of Bz-423 isdistinct from the molecular target(s) of these other compounds.

Example 23 Bz-423 Binds to the OSCP

As part an early group of mechanistic studies of Bz-423, a biotinylatedanalogue was synthesized by replacing the N-methyl group with ahexylaminolinker to which biotin was covalently attached (thismodification did not alter the activity of Bz-423). This molecule wasused to probe a display library of human breast cancer cDNAs(Invitrogen) that are expressed as fusion proteins on the tip of T7phage. Following the screening methods described by Austin andco-workers using biotinylated version of KF506 to identify new FK506binding proteins, the OSCP component of the mitochondrial F₁F₀-ATPasewas identified as a binding protein for Bz-423 (FIG. 1).

To determine if Bz-423 indeed binds to the OSCP and the affinity of theinteraction, human OSCP was overexpressed in E. coli. Titrating asolution of Bz-423 into the OSCP resulted in quenching of the intrinsicprotein fluorescence and afforded a K_(d) of 200±40 nM (FIG. 2). Thebinding of several Bz-423 analogues was also measured and it was foundthat their affinity for the OSCP paralleled their potency in both wholecell cytotoxicity assays as well as ATPase inhibition experiments usingSMP. These data provided cogent evidence that Bz-423 binds to the OSCPon the mitochondrial ATPase. Bz-423 is the only known inhibitor of theATPase that functions through binding to the OSCP. Since the OSCP doesnot contain the ATP binding site and it does not comprise the protonchannel, it is possible that Bz-423 functions by altering the molecularmotions of the ATPase motor.

Example 24 RNAi Knockouts of the OSCP Protect Against Bz-423 InducedCell Death

To complement the chemical and biochemical target identification andvalidation studies described above, experiments were conducted toknockout the OSCP in whole cells. In vitro, removing the OSCP from theATPase abolishes synthase function without altering the hydrolyticactivity of the enzyme. In yeast, OSCP knockouts are not lethal; inthese cells, hydrolysis of ATP provides the chemical potential tosupport ΔΨ_(m) thereby maintaining mitochondrial integrity. Since yeastOSCP has limited sequence homology to the mammalian protein (˜30%),these experiments were conducted in cell lines from human origin.

Since the OSCP is nuclear encoded, RNA interference (RNAi), a techniquethat can achieve post-transcriptional gene silencing, was employed toknockout this protein. For these experiments, HEK 293 cells weretransfected with each of three chemically synthesized small interferingRNA molecules (siRNA) specific for the OSCP sequence usingoligofectamine These cells are transfected in a highly efficient (90%)manner by oligofectamine. OSCP expression was analyzed by immunoblot at24 h, 48 h, 72 h and 96 h after transfection. The maximum silencing ofOSCP expression (64%) occurred at 72 h after transfection (FIG. 3). OSCPsiRNA transfected HEK 293 cells had a reduced Bz-ROS and apoptosis inresponse to Bz-423 relative to cells transfected with a scrambledsequence control siRNA. These results indicated that siRNA is effectiveat reducing OSCP and suggested that Bz-423 mediated cell death signalinginvolves the OSCP.

Example 25 Effect of Bz-423 on Cellular Proliferation

Like most 1,4-benzodiazepines, Bz-423 binds strongly to bovine serumalbumin (BSA), which reduces the effective concentration of drug free insolution. For example, in tissue culture media containing 10% (v/v)fetal bovine serum (FBS), ca. 99% of the drug is bound to BSA.Therefore, cell culture cytotoxicity assays are conducted in media with2% FBS to reduce binding to BSA and increase the free [Bz-423]. Underthese conditions, the dose response-curve is quite sharp such that thereis a limited concentration range at which Bz-423 is only partlyeffective. Since some benzodiazepines are known to haveanti-proliferative properties, the effect of Bz-423 at concentrations<ED₅₀ were carefully analyzed and observed that in addition to inducingapoptosis, Bz-423 prevented cell growth after 3 d in culture. In theselow serum conditions, the cytotoxic and anti-proliferative effectsoverlapped making it difficult to study each effect independently.However, by increasing the [BSA] or increasing FBS to 10%, thedose-response curve flattened (and the cytotoxicity ED₅₀ increased) andBz-423 induced cytotoxicicty could be clearly distinguished from effectson proliferation. At lower amounts of drug (e.g., 10-15 μM), Bz-423 hadminimal cytotoxicity whereas at concentrations >20 μM only apoptosis wasobserved (the death pathway described above including a bimodal ROSresponse, and was also observed in media containing 10% FBS). Whilehigher amounts of drug may also block proliferation, it caused apoptosiswell before the effects on proliferation could be observed. Doseresponse curves were similar in experiments where BSA was added to mediacontaining 2% FBS to simulate media containing 10% FBS, whichdemonstrated that antiproliferation and cytotoxicity were not affectedby other constituents of serum.

To confirm the decrease in cell number relative to control cells after 3d of treatment is due to decreased proliferation and not cell deathbalanced by proliferation, in addition to cell counting, cell divisionswere studied. PKH-67 is a fluorescent probe that binds irreversibly tocell membranes and upon cell division is partitioned equally between thedaughter cells, making it possible to quantify cell division by flowcytometry. Ramos cells stained with PKH67 and treated with Bz-423 hadfewer cell divisions at sub-cytotoxic concentrations which confirmedthat the decrease in cell number was due to anti-proliferative affectsand not cell death. To determine if Bz-423 induced anti-proliferationwas specific to Ramos cells, cell counting and cell cycle experimentswere done in other B cell lines and cell lines derived from solidtumors. As seen in Table 3, the effects on blocking proliferation werenot unique to lymphoid cells which suggested a target, common tomultiple tissue types, mediated the block in proliferation.

TABLE 3 ED₅₀ (μM) for antiproliferation of cells treated for 72 h inmedia with 10% FBS. Cells for study included Ramos cells and clonestransfected to overexpress Bcl-2 and Bcl-x_(L), ovarian cells with nullp53 (SKOV3); neuroblastoma cell lines (IMR-32, Lan-1, SHEP-1); andmalignant B cell lines. Ramos Bcl-2 Bcl-X_(L) SKOV3 IMR-32 Lan-1 SHEP-1CA46 Raji 10.7 11.9 13.7 18.2 18.0 13.7 15.9 13.4 12.9

Example 26 Gene Profiling Cells Treated with Bz-423

Gene profiling experiments were conducted to probe the mechanism bywhich Bz-423 blocks cellular proliferation. In studies usingcyclohexamide as an inhibitor of protein synthesis, it was found thatBz-423-induced cell death did not depend on new protein synthesis.Therefore, changes in gene expression were more likely relevant only tothe mechanism of anti-proliferation. To increase the likelihood ofdetecting changes involved in signal-response coupling rather thandown-stream effects, cells were profiled that were treated with Bz-423for 3 h. This is the point just after the ROS early maximum, but beforeother cellular changes occur, including opening of the mitochondriapermeability pore.

The discovery of the pro-apoptotic, cytotoxic and growth inhibitoryproperties of Bz-423 against pathogenic cell types identified thepotential for this class of agents to be therapeutic against autoimmunediseases, cancers and other neoplastic diseases. Further experimentalevidence from an analysis of the changes in gene expression induced bythis agent expanded the mechanistic understanding of this compound'saction and added to the collection of therapeutic effects it modulates.

In vitro testing with Ramos cells to determine the changes in geneexpression (at the level of mRNA) induced by Bz-423 was performed byculturing cells at a density of 500,000 cells per ml. Solvent control(DMSO, final concentration 0.1% V/V]), Bz-423, or Bz-OMe (10 μM) wasadded to cells. After 4 h, cells were harvested and RNA prepared usingTrizol Reagent (#15596-018, Life Technologies, Rockville, Md.) and theRNeasy Maxi Kit (#75162, Qiagen, Valencia, Calif.) according tomanufacturers protocols. Single stranded cDNA was synthesized by reversetranscription using poly (A) RNA present in the starting total RNAsample. Single stranded cDNA was converted into double stranded cDNA andthen in vitro transcription carried out in the presence of biotinylatedUTP and CTP to produce biotin-labeled cRNA. cRNA was fragmented in thepresence of Mg2+, and hybridized to the human genome U133A Genechiparray (Affymetrix). Hybridization results were quantified using aGeneArray scanner and analysis carried out according to the instructionsprovided by Affymetrix.

Expression profiling using RNA isolated from cells treated with Bz-423,Bz-OMe, or vehicle control was done with the HGU133A Affymetrix genechip, which represents about 22,000 human genes. Using criteria thatinclude p<0.01, 16 genes are expressed 8-fold or more over controlcells. As expected based on the molecular target of Bz-423, many ofthese genes were involved in glycolysis.

The data were analyzed to detect genes changes Bz treatment according tothe criteria that the log-transformed mean signal changed at leastfour-fold in treated compared to vehicle control samples and that thecoefficient of variance for control values (n=4) was less than 10%.These genes represent targets that may mediate therapeutic responses.

The gene expression results for Bz-423 and Bz-OMe each provide a uniquefingerprint of information. The structure of Bz-OMe is as follows:

Expression of some genes change similarly after exposure to both Bz-423and Bz-OMe. Thus, the genes that are commonly regulated between the twocompounds are particularly relevant for understanding gene regulationthrough a more general class of compounds. FIG. 4 presents data showinggene expression profiles of cells treated by Bz-423 and Bz-OMe.

Example 27 Effect of Bz-423 on ODC Levels and Activity

To determine whether ODC activity and polyamine metabolism is affectedby Bz-423, as suggested by RNA profiling data, ODC activity in cellstreated with Bz-423 was directly measured in comparison with a vehiclecontrol. In these experiments, the conversion of ornithine to putrescinewas quantified using ³H-ornithine. For comparisons, control cells weretreated with vehicle control or difluoromethyl ornithine (DFMO), apotent inhibitor of ornithine decarboxylase (like Bz-423, DFMO is apotent anti-proliferative agent). As seen in FIG. 3, treating cells for4 h with Bz-423 significantly reduced ODC activity in a dose-dependantfashion, which is consistent with among other things, an incrrease inantizyme 1, as suggested by RNA profiling. The reduction in ODC activitywas paralleled by a decrease in ODC protein levels measured under thesame conditions.

As described above, Bz-423 induced apoptosis was signaled by an ROSresponse that arose from MRC complex III as a result of the state 3 to 4transition. It was next sought to determine if the ROS response,critical for apoptosis, also mediated these effects on ODC. If the ROSwas required for the decrease in ODC activity, it would likewise beimplicated as potentially part of the anti-proliferative response toBz-423. To test this, Ramos cells were treated with Bz-423, DFMO, orvehicle control for 4 h. In parallel, a second group of cells waspre-incubated with MnTBAP to limit the ROS and then cultured withBz-423, DFMO, and vehicle control. MnTBAP significantly reversedinhibition of ODC by Bz-423.

Collectively these data suggested the possible interpretation that high[Bz-423] (e.g.>10 μM) generate sufficient amounts of ROS that could notbe detoxified by cellular anti-oxidants, and resulted in apoptosiswithin 18 h. Lower [Bz-423] induced a proportionally smaller ROSresponse that was insufficient to trigger apoptosis. In this case,however, the ROS may be capable of inhibiting ODC or otherwise blockingcellular proliferation.

Consistent with this hypothesis, a compound in which the phenolichydroxyl is replaced by Cl (designated Bz-C1) was minimally cytotoxic(activity decreased by ca 80% compared to Bz-423) and generated a smallROS response in cells, while also binding less tightly to the OSCP(K_(d)

5 μM). This compound also inhibited ODC activity (FIG. 3), as predictedby the above hypothesis. Given the proposed role and nature of Bz-423induced ROS in mediating growth arrest, Bz-Cl was tested against thepanel of cells in Table 2 and found that after 3 d it reducedproliferation to a similar extent as Bz-423, with comparable ED₅₀values. These results demonstrated that the antiproliferative effects ofthese compounds could be obtained using chemical analogues of Bz-423that block proliferation without inducing apoptosis.

Example 28 Structure Activity Studies of Novel Cytotoxic Benzodiazepines

Based on these properties of Bz-423, a range of Bz-423 derivatives weresynthesizedto probe structural elements of this novel compound importantfor binding and activity. Replacing the N-methyl group or chlorine witha hydrogen had little effect on lymphotoxic activity againstimmortalized Ramos B cells or Jurkat T cells in culture. Similarly, bothenantiomers of Bz-423 were equipotent, which indicates that theinteraction between Bz-423 and its molecular target involves two-pointbinding. In contrast to these data, removing a naphthalalanine (seeTable 1). The present invention is not limited to a particularmechanism, and an understanding of a mechanism is not necessary topractice the present invention, nonetheless, it is contemplated thatmoiety or replacing the phenolic hydroxyl group with hydrogen abolishedall cytotoxic activity (Table 1). Based on these observations changes tothe C′3 and C′4 positions were investigated. Replacing 1-naphthol with2-naphtho has little effect on cell killing. Similarly, replacing thenapthylalanine with other hydrophobic groups of comparable size hadlittle effect on cytotoxic properties of Bz-423. By contrast, quinolines7-9 were each less potent than Bz-423. The present invention is notlimited to a particular mechanism, and an understanding of a mechanismis not necessary to practice the present invention, nonetheless, it iscontemplated that theses data suggest a preference for a hydrophobicsubstituent within the binding site for Bz-423. Smaller C3 substituentswere only somewhat less potent than Bz-423 whereas compounds witharomatic groups containing oxygen were significantly less cytotoxic.These data clearly indicate that a bulky hydrophobic aromaticsubstituent is useful for optimal activity.

TABLE 1 Potency of Bz-423 derivatives. Cell death was assessed byculturing Ramos B cells in the presence of each compound in adose-response fashion. Cell viability was measured after 24 h propidiumiodide exclusion using flow cytometry. In this assay, the EC₅₀ forPK11195, diazepam, and 4-Cl-diazepam is >80 μM. Compound EC₅₀ (μM)_(a)-naphthalAla 1 >80 -phenol 2 >80

3 5

4 4

5 7

6 4

7 11

8 12

9 15 Compound EC₅₀ (μM)

10 12

11 10

12 6

13 7

14 35

15 25 _(a)Each EC₅₀ value was determined twice in triplicate and has anerror of ±5%.

Placing a methyl group ortho to the hydroxyl (16) does not alter theactivity of Bz-423 whereas moving the hydroxyl to the C′4 (17) positiondecreased potency 2-fold (Table 2). By contrast, replacing the hydroxylwith chlorine or azide, or methylating the phenol effectively abolishesthe cytotoxic activity of Bz-423. The present invention is not limitedto a particular mechanism, and an understanding of the mechanism is notnecessary to practice the present invention, nonetheless, it iscontemplated that these data indicate that a hydroxyl group positionedat the C′4 carbon is required for optimal activity, possibly by making acritical contact upon target binding. However, molecules possessing aphenolic substructure can also act as alternate electron carriers withinthe MRC. Such agents accept an electron from MRC enzymes and transfer itback to the chain at point of higher reducing potential. This type of‘redox cycling’ consumes endogenous reducing equivalents (e.g.,glutathione) along with pyrimidine nucleotides and results in celldeath. To distinguish between these alternatives, it was determinedwhether Bz-423 redox cycles in the presence of sub-mitochondrialparticles using standard NADH and NAD(P)H oxidation assays. Unlike thepositive controls (doxorubicin and menadione), Bz-423 does not lead tosubstrate oxidation which strongly suggests that it does not redoxcycle. The present invention is not limited to a particular mechanism,and an understanding of the mechanism is not necessary to practice thepresent invention, nonetheless, it is contemplated that collectively,the data indicate that the decreased activity of compounds 18-20 resultsfrom removing an interaction that mediates binding of Bz-423 to itstarget protein.

TABLE 2 Potency of Bz-423 derivatives. Cell death was assessed asdescribed in Table 1

Compound 16 17 18 19 20 EC₅₀ 3 6 >80 >80 >80

Cells rapidly produce O₂ ⁻ in response to Bz-423 and blocking thissignal (e.g., by inhibiting ubiquinol cytochrome c reductase, which isthe enzyme that produces O₂ ⁻ in response to Bz-423) prevents apoptosis.To determine if the Bz-423 derivatives kill cells in manner analogous toBz-423 (presumably as a result of binding to a common molecular target),the ability of FK506 was examined, micromolar amounts of whicheffectively inhibit ubiquinol cytochrome c reductase, to protect againstcell death Inhibition by FK506 (

60%) was only observed for 3-6, 12, 13, 16, and 17, which are thecompounds with hydrophobic C3 side chains larger than benzene. Celldeath induced by each of these compounds (including Bz-423) was alsoinhibited (to

60%) by pre-treating cells with either 18, 19, or 20 (at >40 μM).Compounds 18, 19, and 20 had no effect on blocking the cytotoxicactivity (inhibition of

20%) of the other benzodiazepines listed in Table 2. The presentinvention is not limited to a particular mechanism, and an understandingof the mechanism is not necessary to practice the present invention,nonetheless, it is contemplated that these data strongly suggest thatBz-423 along with 3-6, 12, 13, 16, and 17 bind the same site within thetarget protein and induce apoptosis through a common mechanism. Theother compounds do not bind at this site and induce a death responsethrough a different pathway.

All publications and patents mentioned in the above specification areherein incorporated by reference. Although the invention has beendescribed in connection with specific preferred embodiments, it shouldbe understood that the invention as claimed should not be unduly limitedto such specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention that are obvious to thoseskilled in the relevant fields are intended to be within the scope ofthe following claims.

We claim:
 1. A compound represented by Formula I:

or a pharmaceutically acceptable salt thereof; wherein R₁ is alkyl; R₂is halogen; R₃ and R₄ are hydrogen; and R₅ is

 and the stereochemical configuration at a stereocenter in said compoundis R, S, or a mixture thereof.
 2. The compound or pharmaceuticallyacceptable salt thereof of claim 1, wherein the stereochemicalconfiguration at the stereocenter of the compound is (R).
 3. Thecompound or pharmaceutically acceptable salt thereof of claim 1, whereinthe stereochemical configuration at the stereocenter of the compound is(S).
 4. A pharmaceutical composition comprising a compound orpharmaceutically acceptable salt thereof of any one of claim 1, 2, or 3and a pharmaceutically acceptable carrier.
 5. A compound represented byFormula I:

wherein: R₁ is alkyl; R₂ is halogen; R₃ and R₄ are hydrogen; and R₅ is

 and the stereochemical configuration at a stereocenter in said compoundis R, S, or a mixture thereof.
 6. The compound of claim 5, wherein thestereochemical configuration at the stereocenter of the compound is (R).7. The compound of claim 5, wherein the stereochemical configuration atthe stereocenter of the compound is (S).
 8. A pharmaceutical compositioncomprising a compound of any one of claim 5, 6, or 7 and apharmaceutically acceptable carrier.
 9. A compound represented byFormula I in the form of a pharmaceutically acceptable salt:

wherein: R₁ is alkyl; R₂ is halogen; R₃ and R₄ are hydrogen; and R₅ is

 and the stereochemical configuration at a stereocenter in said compoundis R, S, or a mixture thereof.
 10. The pharmaceutically acceptable saltof claim 9, wherein the stereochemical configuration at the stereocenterof the compound is (R).
 11. The pharmaceutically acceptable salt ofclaim 9, wherein the stereochemical configuration at the stereocenter ofthe compound is (S).
 12. A pharmaceutical composition comprising apharmaceutically acceptable salt of any one of claim 9, 10, or 11 and apharmaceutically acceptable carrier.